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Sample records for production purification crystallization

  1. 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.

  2. Production, purification, crystallization and preliminary X-ray analysis of adeno-associated virus serotype 8

    SciTech Connect

    Lane, Michael Douglas; Nam, Hyun-Joo; Padron, Eric; Gurda-Whitaker, Brittney; Kohlbrenner, Eric; Aslanidi, George; Byrne, Barry; McKenna, Robert; Muzyczka, Nicholas; Zolotukhin, Sergei; Agbandje-McKenna, Mavis

    2005-06-01

    The production, purification, crystallization and preliminary X-ray crystallographic analysis of adeno-associated virus serotype 8 is reported. Adeno-associated viruses (AAVs) are actively being developed for clinical gene-therapy applications and the efficiencies of the vectors could be significantly improved by a detailed understanding of their viral capsid structures and the structural determinants of their tissue-transduction interactions. AAV8 is ∼80% identical to the more widely studied AAV2, but its liver-transduction efficiency is significantly greater than that of AAV2 and other serotypes. The production, purification, crystallization and preliminary X-ray crystallographic analysis of AAV8 viral capsids are reported. The crystals diffract X-rays to 3.0 Å resolution using synchrotron radiation and belong to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = 257.5, c = 443.5 Å. The unit cell contains two viral particles, with ten capsid viral protein monomers per crystallographic asymmetric unit.

  3. Production, purification and crystallization of a trans-sialidase from Trypanosoma vivax.

    PubMed

    Haynes, Carole L F; Ameloot, Paul; Remaut, Han; Callewaert, Nico; Sterckx, Yann G J; Magez, Stefan

    2015-05-01

    Sialidases and trans-sialidases play important roles in the life cycles of various microorganisms. These enzymes can serve nutritional purposes, act as virulence factors or mediate cellular interactions (cell evasion and invasion). In the case of the protozoan parasite Trypanosoma vivax, trans-sialidase activity has been suggested to be involved in infection-associated anaemia, which is the major pathology in the disease nagana. The physiological role of trypanosomal trans-sialidases in host-parasite interaction as well as their structures remain obscure. Here, the production, purification and crystallization of a recombinant version of T. vivax trans-sialidase 1 (rTvTS1) are described. The obtained rTvTS1 crystals diffracted to a resolution of 2.5 Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 57.3, b = 78.4, c = 209.0 Å.

  4. 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.

  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. 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).

  7. Water Purification Product

    NASA Technical Reports Server (NTRS)

    2004-01-01

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

  8. Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

    PubMed

    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

    1995-10-01

    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.

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

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    2005-08-01

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

  10. Renaissance of protein crystallization and precipitation in biopharmaceuticals purification.

    PubMed

    Dos Santos, Raquel; Carvalho, Ana Luísa; Roque, A Cecília A

    The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups.

  11. Production, purification and properties of microbial phytases.

    PubMed

    Pandey, A; Szakacs, G; Soccol, C R; Rodriguez-Leon, J A; Soccol, V T

    2001-05-01

    Phytases (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) catalyse the release of phosphate from phytate (mycoinositol hexakiphosphate). Several cereal grains, legumes and oilseeds, etc., store phosphorus as phytate. Environmental pollution due to the high-phosphate manure, resulting in the accumulation of P at various locations has raised serious concerns. Phytases appear of significant value in effectively controlling P pollution. They can be produced from a host of sources including plants, animals and micro-organisms. Microbial sources, however, are promising for their commercial exploitations. Strains of Aspergillus sp., chiefly A. ficuum and A. niger have most commonly been employed for industrial purposes. Phytases are considered as a monomeric protein, generally possessing a molecular weight between 40 and 100 kDa. They show broad substrate specificity and have generally pH and temperature optima around 4.5-6.0 and 45-60 degrees C. The crystal structure of phytase has been determined at 2.5 A resolution. Immobilization of phytase has been found to enhance its thermostability. This article reviews recent trends on the production, purification and properties of microbial phytases.

  12. Protein Purification and Its Application to Crystallization

    DTIC Science & Technology

    1988-08-30

    4-1 4.1.2.2 Microcystin ...............................4-5 4.1.2.3 Aequorin.........................4-5 4.1.2.4 Staphylococcal...analyzing these crystals by HPLC to determine crystal composition. Crystals of microcystin , which did not diffract, were isolated from a hanging drop...standard curve was prepared to correlate peak height to micrograms of microcystin injected. The results of this experiment showed that the crystals did

  13. 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.

  14. 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.

  15. 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.

  16. Purification and crystallization of oxygen-evolving photosystem II core complex from thermophilic cyanobacteria.

    PubMed

    Shen, Jian-Ren; Kawakami, Keisuke; Koike, Hiroyuki

    2011-01-01

    This chapter describes the purification and crystallization of oxygen-evolving photosystem II core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus. Procedures used for purification of photosystem II from the cyanobacterium involves cultivation of cells, isolation of thylakoid membranes, purification of crude and pure photosystem II core complexes by detergent solubilization, followed by differential centrifugation and column chromatography. The purified core dimer particles were successfully used for crystallization, and the methods and conditions used for crystallization are presented. These purification and crystallization procedures can be applied for another thermophilic cyanobacterium T. elongatus.

  17. Purification of Germanium Crystals by Zone Refining

    NASA Astrophysics Data System (ADS)

    Kooi, Kyler; Yang, Gang; Mei, Dongming

    2016-09-01

    Germanium zone refining is one of the most important techniques used to produce high purity germanium (HPGe) single crystals for the fabrication of nuclear radiation detectors. During zone refining the impurities are isolated to different parts of the ingot. In practice, the effective isolation of an impurity is dependent on many parameters, including molten zone travel speed, the ratio of ingot length to molten zone width, and number of passes. By studying the theory of these influential factors, perfecting our cleaning and preparation procedures, and analyzing the origin and distribution of our impurities (aluminum, boron, gallium, and phosphorous) identified using photothermal ionization spectroscopy (PTIS), we have optimized these parameters to produce HPGe. We have achieved a net impurity level of 1010 /cm3 for our zone-refined ingots, measured with van der Pauw and Hall-effect methods. Zone-refined ingots of this purity can be processed into a detector grade HPGe single crystal, which can be used to fabricate detectors for dark matter and neutrinoless double beta decay detection. This project was financially supported by DOE Grant (DE-FG02-10ER46709) and the State Governor's Research Center.

  18. Biopharmaceuticals from microorganisms: from production to purification.

    PubMed

    Jozala, Angela Faustino; Geraldes, Danilo Costa; Tundisi, Louise Lacalendola; Feitosa, Valker de Araújo; Breyer, Carlos Alexandre; Cardoso, Samuel Leite; Mazzola, Priscila Gava; Oliveira-Nascimento, Laura de; Rangel-Yagui, Carlota de Oliveira; Magalhães, Pérola de Oliveira; Oliveira, Marcos Antonio de; Pessoa, Adalberto

    2016-12-01

    The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, β, and γ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

  19. Purification and two-dimensional crystallization of bacterial cytochrome oxidases.

    PubMed

    Warne, A; Wang, D N; Saraste, M

    1995-12-01

    A novel strategy which employes chromatography on an immobilized metal ion has been developed for the purification of bacterial cytochrome c and quinol oxidases. Many bacterial oxidase complexes appear to have a natural affinity to bind to the chelated copper ion. A combination of three different chromatographic principles (anion exchange, metal-affinity and gel filtration) makes an effective tool chest for the preparation of homogeneous and protein-chemically pure bacterial oxidases. These preparations have been used for two-dimensional crystallization. Until now, crystals have been obtained using the Paracococcus denitrificans and Rhodobacter sphaeroides cytochrome aa3 and the Escherichia coli cytochrome bo. The crystals diffract to approximately 2.5 nm in negative stain and have potential for further structural studies.

  20. 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…

  1. Purification of FGD gypsum product

    SciTech Connect

    Rogers, K.J.; Owens, F.C. II.

    1993-06-01

    A method of purifying a gypsum slurry resulting from an upstream flue gas desulfurization process is described comprising the steps of: (a) delivering the gypsum slurry to a primary dewatering device; (b) separating this gypsum slurry in said primary dewatering device into a coarse solids stream and a fine solids stream, said coarse solids stream primarily containing coarse particle sizes therein and said fine solids stream primarily containing fine particle sizes therein; (c) selectively returning all or a portion of said fine solids stream back to the upstream flue gas desulfurization process or delivering all or a portion of said fine solids stream to downstream separation means for further separation into a thickened fines stream and a process water stream, said process water stream thereafter being selectively delivered, as desired, to the upstream flue gas desulfurization process; (d) delivering said coarse solids stream to a surge/mix tank where it is selectively mixed with a portion of said thickened fines stream prior to being delivered to a secondary dewatering device; and, (e) collecting a purified gypsum product from said secondary dewatering device.

  2. 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.

  3. Isolation and Purification of Biotechnological Products

    NASA Astrophysics Data System (ADS)

    Hubbuch, Jürgen; Kula, Maria-Regina

    2007-05-01

    The production of modern pharma proteins is one of the most rapid growing fields in biotechnology. The overall development and production is a complex task ranging from strain development and cultivation to the purification and formulation of the drug. Downstream processing, however, still accounts for the major part of production costs. This is mainly due to the high demands on purity and thus safety of the final product and results in processes with a sequence of typically more than 10 unit operations. Consequently, even if each process step would operate at near optimal yield, a very significant amount of product would be lost. The majority of unit operations applied in downstream processing have a long history in the field of chemical and process engineering; nevertheless, mathematical descriptions of the respective processes and the economical large-scale production of modern pharmaceutical products are hampered by the complexity of the biological feedstock, especially the high molecular weight and limited stability of proteins. In order to develop new operational steps as well as a successful overall process, it is thus a necessary prerequisite to develop a deeper understanding of the thermodynamics and physics behind the applied processes as well as the implications for the product.

  4. 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

  5. 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.

  6. 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.

  7. 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.

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

    DOE PAGES

    Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi; ...

    2014-08-20

    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 tomore » 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.« less

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Marmont, Lindsey S.; Whitney, John C.; Robinson, Howard; Colvin, Kelly M.; Parsek, Matthew R.; Howell, P. Lynne

    2012-01-01

    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 Å, α = β = γ = 90.0°. The PelD crystals exhibited the symmetry of space group P21212 and diffracted to a minimum d-spacing of 2.2 Å. On the basis of the Matthews coefficient (V M = 2.29 Å3 Da−1), it was estimated that two molecules are present in the asymmetric unit. PMID:22297994

  12. Production and partial purification of Salmonella enterotoxin.

    PubMed Central

    Sedlock, D M; Koupal, L R; Deibel, R H

    1978-01-01

    By using a strain of Salmonella typhimurium, we detected the presence of an enterotoxin, as determined by the rabbit ileal loop assay, in various complex and defined media. The enterotoxin was concentrated by ultrafiltration of culture supernatant fluids and eluted in and adjacent to the void volume of a Sephadex G-100 column. This suggested that the enterotoxic factor was of a relatively high molecular weight, and additional evidence indicated it was heterogeneous in size. Further chromatography, using a diethylaminoethyl-cellulose anion exchanger, facilitated at least a 50-fold purification of the Salmonella enterotoxin. PMID:352941

  13. 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

  14. Production and purification of the multifunctional enzyme horseradish peroxidase

    PubMed Central

    Spadiut, Oliver; Herwig, Christoph

    2014-01-01

    The oxidoreductase horseradish peroxidase (HRP) is used in numerous industrial and medical applications. In this review, we briefly describe this well-studied enzyme and focus on its promising use in targeted cancer treatment. In combination with a plant hormone, HRP can be used in specific enzyme–prodrug therapies. Despite this outstanding application, HRP has not found its way as a biopharmaceutical into targeted cancer therapy yet. The reasons therefore lie in the present low-yield production and cumbersome purification of this enzyme from its natural source. However, surface glycosylation renders the recombinant production of HRP difficult. Here, we compare different production hosts for HRP and summarize currently used production and purification strategies for this enzyme. We further present our own strategy of glycoengineering this powerful enzyme to allow recombinant high-yield production in Pichia pastoris and subsequent simple downstream processing. PMID:24683473

  15. (Hyper)thermophilic enzymes: production and purification.

    PubMed

    Falcicchio, Pierpaolo; Levisson, Mark; Kengen, Servé W M; Koutsopoulos, Sotirios

    2014-01-01

    The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

  16. 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.

  17. 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…

  18. Purification of Sewage Contaminated by Oil Products Using Mesoporous Coal

    NASA Astrophysics Data System (ADS)

    Gvazava, Elene; Maisuradze, Nino; Samkharadze, Irma

    2016-10-01

    The sorption properties of mesoporous coals (pore size of ∼⃒ 4 nm, the specific surface area of 25 to 150 m2/g) of Georgian hard coal deposit have been studied and the efficacy of their usage for the treatment of sewage water polluted by oil products has been established. Purification rate depends on coal mass loaded in filter, grain size, initial concentration of oil products, the water acidity, etc.

  19. Recent advances in production, purification and applications of phycobiliproteins.

    PubMed

    Sonani, Ravi Raghav; Rastogi, Rajesh Prasad; Patel, Rutvij; Madamwar, Datta

    2016-02-26

    An obligatory sunlight requirement for photosynthesis has exposed cyanobacteria to different quantity and quality of light. Cyanobacteria can exhibit efficient photosynthesis over broad region (450 to 650 nm) of solar spectrum with the help of brilliantly coloured pigment proteins called phycobiliproteins (PBPs). Besides light-harvesting, PBPs are found to involve in several life sustaining phenomena including photoprotection in cyanobacteria. The unique spectral features (like strong absorbance and fluorescence), proteineous nature and, some imperative properties like hepato-protective, anti-oxidants, anti-inflammatory and anti-aging activity of PBPs enable their use in food, cosmetics, pharmaceutical and biomedical industries. PBPs have been also noted to show beneficial effect in therapeutics of some disease like Alzheimer and cancer. Such large range of applications increases the demand of PBPs in commodity market. Therefore, the large-scale and coast effective production of PBPs is the real need of time. To fulfil this need, many researchers have been working to find the potential producer of PBPs for the production and purification of PBPs. Results of these efforts have caused the inventions of some novel techniques like mixotrophic and heterotrophic strategies for production and aqueous two phase separation for purification purpose. Overall, the present review summarises the recent findings and identifies gaps in the field of production, purification and applications of this biological and economically important proteins.

  20. Recent advances in production, purification and applications of phycobiliproteins

    PubMed Central

    Sonani, Ravi Raghav; Rastogi, Rajesh Prasad; Patel, Rutvij; Madamwar, Datta

    2016-01-01

    An obligatory sunlight requirement for photosynthesis has exposed cyanobacteria to different quantity and quality of light. Cyanobacteria can exhibit efficient photosynthesis over broad region (450 to 650 nm) of solar spectrum with the help of brilliantly coloured pigment proteins called phycobiliproteins (PBPs). Besides light-harvesting, PBPs are found to involve in several life sustaining phenomena including photoprotection in cyanobacteria. The unique spectral features (like strong absorbance and fluorescence), proteineous nature and, some imperative properties like hepato-protective, anti-oxidants, anti-inflammatory and anti-aging activity of PBPs enable their use in food, cosmetics, pharmaceutical and biomedical industries. PBPs have been also noted to show beneficial effect in therapeutics of some disease like Alzheimer and cancer. Such large range of applications increases the demand of PBPs in commodity market. Therefore, the large-scale and coast effective production of PBPs is the real need of time. To fulfil this need, many researchers have been working to find the potential producer of PBPs for the production and purification of PBPs. Results of these efforts have caused the inventions of some novel techniques like mixotrophic and heterotrophic strategies for production and aqueous two phase separation for purification purpose. Overall, the present review summarises the recent findings and identifies gaps in the field of production, purification and applications of this biological and economically important proteins. PMID:26981199

  1. 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.

  2. American cranberry products: proanthocyanidin purification and concentrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    American cranberry (Vaccinium macrocarpon Ait.) phenolics have important roles within the plant; they also contribute to harvest and product quality, and have potential human health benefits. Proanthocyanidins (phenolic polymers) may aid in preventing urinary tract infections (UTIs), although litera...

  3. 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.

  4. Improvements in G protein-coupled receptor purification yield light stable rhodopsin crystals.

    PubMed

    Salom, David; Le Trong, Isolde; Pohl, Ehmke; Ballesteros, Juan A; Stenkamp, Ronald E; Palczewski, Krzysztof; Lodowski, David T

    2006-12-01

    G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins and are the target of approximately half of all therapeutic agents. Agonist ligands bind their cognate GPCRs stabilizing the active conformation that is competent to bind G proteins, thus initiating a cascade of intracellular signaling events leading to modification of the cell activity. Despite their biomedical importance, the only known GPCR crystal structures are those of inactive rhodopsin forms. In order to understand how GPCRs are able to transduce extracellular signals across the plasma membrane, it is critical to determine the structure of these receptors in their ligand-bound, active state. Here, we report a novel combination of purification procedures that allowed the crystallization of rhodopsin in two new crystal forms and can be applicable to the purification and crystallization of other membrane proteins. Importantly, these new crystals are stable upon photoactivation and the preliminary X-ray diffraction analysis of both photoactivated and ground state rhodopsin crystals are also reported.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Matrix product purifications for canonical ensembles and quantum number distributions

    NASA Astrophysics Data System (ADS)

    Barthel, Thomas

    2016-09-01

    Matrix product purifications (MPPs) are a very efficient tool for the simulation of strongly correlated quantum many-body systems at finite temperatures. When a system features symmetries, these can be used to reduce computation costs substantially. It is straightforward to compute an MPP of a grand-canonical ensemble, also when symmetries are exploited. This paper provides and demonstrates methods for the efficient computation of MPPs of canonical ensembles under utilization of symmetries. Furthermore, we present a scheme for the evaluation of global quantum number distributions using matrix product density operators (MPDOs). We provide exact matrix product representations for canonical infinite-temperature states, and discuss how they can be constructed alternatively by applying matrix product operators to vacuum-type states or by using entangler Hamiltonians. A demonstration of the techniques for Heisenberg spin-1 /2 chains explains why the difference in the energy densities of canonical and grand-canonical ensembles decays as 1 /L .

  12. 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.

  13. 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.

  14. 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.

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

    PubMed

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

    2007-09-01

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

  16. Recent insights into microbial catalases: isolation, production and purification.

    PubMed

    Sooch, Balwinder Singh; Kauldhar, Baljinder Singh; Puri, Munish

    2014-12-01

    Catalase, an oxidoreductase enzyme, works as a detoxification system inside living cells against reactive oxygen species formed as a by-product of different metabolic reactions. The enzyme is found in a wide range of aerobic and anaerobic organisms. Catalase has also been employed in various analytical and diagnostic methods in the form of biosensors and biomarkers in addition to its other applications in textile, paper, food and pharmaceutical industries. New applications for catalases are constantly emerging thanks to their high turnover rate, distinct evolutionary origin, relatively simple and well-defined reaction mechanisms. The following review provides comprehensive information on isolation, production and purification of catalases with different techniques from various microbial sources along with their types, structure, mechanism of action and applications.

  17. 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.

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

    PubMed

    Sun, Lifang; Wu, Yunkun

    2016-08-01

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

  19. Production and Purification of Bioethanol from Molasses and Cassava

    NASA Astrophysics Data System (ADS)

    Maryana, Roni; Wahono, Satriyo Krido

    2009-09-01

    This research aim to analysis bioethanol purification process. Bioethanol from cassava has been produced in previous research and the ethanol from molasses was taken from Bekonang region. The production of bioethanol from cassava was carried out through several processes such as homogenization, adding of α-amylase, β-amylase and yeast (Saccharomyces c). Two types of laboratory scale distillator have been used, the first type is 50 cm length and 4 cm diameter. The second type distillator is 30 cm length and 9 cm diameter. Both types have been used to distill bioethanol The initial concentration after the fermentation process is 15% for bioethanol from cassava and 20-30% ethanol from molasses. The results of first type distillator are 90% of bioethanol at 50° C and yield 2.5%; 70% of bioethanol at 60° C and yield 11.2%. 32% of bioethanol at 70° C and yield 42%. Meanwhile the second distillator results are 84% of bioethanol at 50° C with yield 12%; 51% of bioethanol at 60° C with yield 35.5%; 20% of bioethanol at 70° C with yield 78.8%; 16% of bioethanol at 80° C with yield 81.6%. The ethanol from molasses has been distillated once times in Bekonang after the fermentation process, the yield was about 20%. In this research first type distillator and the initial concentration is 20% has been used. The results are 95% of bioethanol at 75° C with yield 8%; 94% of bioethanol at 85° C with yield 13% when vacuum pump was used. And 94% of bioethanol at 90° C with yield 3.7% and 94% of bioethanol at 96° C with yield 10.27% without vacuum pump. The bioethanol purification use second type distillator more effective than first type distillator.

  20. 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.

  1. [Assessment of schemes for sewage purification from petroleum products, by using various flotation methods].

    PubMed

    Zabuga, G A; Filippova, T M; Sivkov, A A

    2010-01-01

    Petroleum products are the most common pollutants in petroleum refinery wastewater and are freed from the latter by flotation that is one of the most frequently applied physicochemical methods. The existing petroleum refinery OAO "Angara Petroleum Company" scheme for sewage purification from petroleum products, by using pressure flotation and proposed as a competitive purification scheme by applying electrical and impeller flotations underwent a comparative ecologoeconomic analysis. The use of electrical flotation instead of pressure flotation and that of an impeller flotation-electrical flotation system instead of a mechanical purification-pressure flotation one can considerably lower the concentration of petroleum products at the wastewater outlet into the Angara river.

  2. 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.

  3. 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.

  4. 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.

  5. High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins.

    PubMed

    Gabrielsen, Mads; Kroner, Frank; Black, Isobel; Isaacs, Neil W; Roe, Andrew J; McLuskey, Karen

    2011-01-01

    An important factor in the crystallization, and subsequent structural determination, of integral membrane proteins is the ability to produce a stable and monodisperse solution of the protein. Obtaining the correct purification detergent to achieve this can be laborious and is often serendipitous. In this study, high-throughput methods are used to analyze the suitability of eight different detergents on the stability of 12 inner transmembrane proteins from Escherichia coli. The best results obtained from the small-scale experiments were scaled up, the aggregation state of the proteins assessed, and all monodisperse protein solutions entered into crystallization trials. This resulted in preliminary crystallization hits for three inner membrane proteins: XylH, PgpB and YjdL and this study reports the methods, purification procedures and crystallization conditions used to achieve this.

  6. Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure.

    PubMed

    Brazzolotto, Xavier; Wandhammer, Marielle; Ronco, Cyril; Trovaslet, Marie; Jean, Ludovic; Lockridge, Oksana; Renard, Pierre-Yves; Nachon, Florian

    2012-08-01

    Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ≈ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 Å. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.

  7. 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.

  8. 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.

  9. Final Scientific/Technical Report for "A Novel,Highly Efficient and Economic Purification Process Revolutionizing PTA Production"

    SciTech Connect

    Wytcherley, Randi; Balderston, Kristen; Ball,George; Chou, Tai-Li

    2008-06-06

    GTC Technology, Inc., under a cooperative agreement with the DOE’s Industrial Technologies Program and in collaboration with Montana State University, has completed pilot scale testing of a revolutionary new process to produce purified terephthalic acid (PTA), a crucial chemical commodity manufactured worldwide. Purified terephthalic acid (PTA) is a starting material for the formation of polyester resin. Polyester resin is used to make many valuable commercial products, including clothing, plastic containers and films. In traditional PTA production, critical reactions are carried out at high temperatures and pressures, creating physically harsh and economically costly operating conditions. The chemical halide bromine is an essential ingredient for part of the conventional process. As a result of using bromine, the highly toxic and environmentally insidious compound methyl bromide is formed. The corrosive nature of bromine also mandates the use of specialized and expensive corrosion-resistant materials for plant construction. Plants processing PTA conventionally must also use copious amounts of precious water resources and manage costly water treatment operations. GTC’s new TA purification method employs a unique two-step crystallization process capable of operating at lower temperatures and pressures than traditional methods. Utilization of a highly selective, proprietary organic solvent blend also allows for the flexibility of accepting higher levels of impurities in the initial purification feedstock. The relaxed physical operating conditions combined with the effectiveness of the blended organic solvent allow for the efficient purification of feedstock created in a bromine free manner. Along with the elimination of bromine, the new purification technology drastically reduces energy costs and expensive wastewater treatment. Industry wide implementation in the United States alone could yield energy savings of 22 trillion BTU per year. In 2007, using the

  10. 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.

  11. Purification, crystallization and preliminary X-ray analysis of phycocyanin and phycoerythrin from Porphyra yezoensis Ueda.

    PubMed

    Cai, Chuner; Wu, Lian; Li, Chunxia; He, Peimin; Li, Jie; Zhou, Jiahai

    2011-05-01

    Porphyra yezoensis is one of the most important and widely cultured seaweeds in China. Phycobiliproteins exhibit excellent spectroscopic properties and play versatile roles in the biomedical, food, cosmetics and chemical synthetic dye industries. Here, the purification and crystallization of phycoerythrin and phycocyanin, two phycobiliproteins extracted from P. yezoensis, are described. Using a novel protocol including co-precipitation with ammonium sulfate and hydroxyapatite column chromatography, both phycobiliproteins were produced on a large scale with improved quality and yield compared with those previously reported. Native PAGE analysis indicated that phycoerythrin and phycocyanin exist as (αβ)(3) heterohexamers in solution. The crystals of phycoerythrin diffracted to 2.07 Å resolution and belonged to space group R3. The unit-cell parameters referred to hexagonal axes are a = b = 187.7, c = 59.7 Å, with nine (αβ)(2) heterotetramers per unit cell. The crystals of phycocyanin diffracted to 2.70 Å resolution in space group P2(1). Matthews coefficient analysis shows that 10-19 (αβ) heterodimers of phycocyanin in the asymmetric unit would be reasonable. A self-rotation function calculation clarified this ambiguity and indicated that 12 (αβ) heterodimers of phycocyanin are assembled in the asymmetric unit.

  12. LARGE SCALE METHOD FOR THE PRODUCTION AND PURIFICATION OF CURIUM

    DOEpatents

    Higgins, G.H.; Crane, W.W.T.

    1959-05-19

    A large-scale process for production and purification of Cm/sup 242/ is described. Aluminum slugs containing Am are irradiated and declad in a NaOH-- NaHO/sub 3/ solution at 85 to 100 deg C. The resulting slurry filtered and washed with NaOH, NH/sub 4/OH, and H/sub 2/O. Recovery of Cm from filtrate and washings is effected by an Fe(OH)/sub 3/ precipitation. The precipitates are then combined and dissolved ln HCl and refractory oxides centrifuged out. These oxides are then fused with Na/sub 2/CO/sub 3/ and dissolved in HCl. The solution is evaporated and LiCl solution added. The Cm, rare earths, and anionic impurities are adsorbed on a strong-base anfon exchange resin. Impurities are eluted with LiCl--HCl solution, rare earths and Cm are eluted by HCl. Other ion exchange steps further purify the Cm. The Cm is then precipitated as fluoride and used in this form or further purified and processed. (T.R.H.)

  13. 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.

  14. Production, purification, and capsid stability of rhinovirus C types.

    PubMed

    Griggs, Theodor F; Bochkov, Yury A; Nakagome, Kazuyuki; Palmenberg, Ann C; Gern, James E

    2015-06-01

    The rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification.

  15. Flipping Crystals Leads to Better Solar Products

    SciTech Connect

    Mohite, Aditya; Nie, Wanyi; Tsai, Hsinnha; Blancon, Jean-Christophe

    2016-07-06

    In a step that could bring perovskite crystals closer to use in the burgeoning solar power industry, researchers from a joint Los Alamos National Laboratory, Northwestern University and Rice University research study have tweaked their crystal production method and developed a new type of 2-dimensional layered perovskite with outstanding stability and more than triple the material’s previous power conversion efficiency.

  16. Use of Escherichia coli for the production and purification of membrane proteins.

    PubMed

    Postis, Vincent G L; Rawlings, Andrea E; Lesiuk, Amelia; Baldwin, Stephen A

    2013-01-01

    Individual types of ion channels and other membrane proteins are typically expressed only at low levels in their native membranes, rendering their isolation by conventional purification techniques difficult. The heterologous over-expression of such proteins is therefore usually a prerequisite for their purification in amounts suitable for structural and for many functional investigations. The most straightforward expression host, suitable for prokaryote membrane proteins and some proteins from eukaryotes, is the bacterium Escherichia coli. Here we describe the use of this expression system for production of functionally active polytopic membrane proteins and methods for their purification by affinity chromatography in amounts up to tens of milligrams.

  17. Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

    PubMed Central

    Marchot, P.; Ravelli, R. B.; Raves, M. L.; Bourne, Y.; Vellom, D. C.; Kanter, J.; Camp, S.; Sussman, J. L.; Taylor, P.

    1996-01-01

    A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions. PMID:8845756

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

    SciTech Connect

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

    2016-08-09

    The gene encoding a putative siderophore-interacting protein from the marine bacterium S. frigidimarina was successfully cloned, followed by expression and purification of the gene product. Optimized crystals diffracted to 1.35 Å resolution and preliminary crystallographic analysis is promising with respect to structure determination and increased insight into the poorly understood molecular mechanisms underlying iron acquisition. 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 P2{sub 1}, 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.

  19. 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.

  20. Recovery and purification process development for monoclonal antibody production

    PubMed Central

    Ma, Junfen; Winter, Charles; Bayer, Robert

    2010-01-01

    Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

  1. 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.

  2. Flipping Crystals Leads to Better Solar Products

    ScienceCinema

    Mohite, Aditya; Nie, Wanyi; Tsai, Hsinnha; Blancon, Jean-Christophe

    2016-07-20

    In a step that could bring perovskite crystals closer to use in the burgeoning solar power industry, researchers from a joint Los Alamos National Laboratory, Northwestern University and Rice University research study have tweaked their crystal production method and developed a new type of 2-dimensional layered perovskite with outstanding stability and more than triple the material’s previous power conversion efficiency.

  3. Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

    PubMed Central

    Iourin, Oleg; Harlos, Karl; El Omari, Kamel; Lu, Weixian; Kadlec, Jan; Iqbal, Munir; Meier, Christoph; Palmer, Andrew; Jones, Ian; Thomas, Carole; Brownlie, Joe; Grimes, Jonathan M.; Stuart, David I.

    2013-01-01

    Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress. PMID:23295482

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

    PubMed

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

    2006-11-01

    Alpha-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 alpha-11 giardin under two different conditions and in two different space groups is reported. Crystals from the first condition diffracted to 1.1 A 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 A and belong to a primitive monoclinic space group. Determination of the detailed atomic structure of alpha-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. 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.

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

    PubMed

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

    2014-11-01

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

  7. Solid olive waste in environmental cleanup: oil recovery and carbon production for water purification.

    PubMed

    El-Hamouz, Amer; Hilal, Hikmat S; Nassar, Nashaat; Mardawi, Zahi

    2007-07-01

    A potentially-economic three-fold strategy, to use solid olive wastes in water purification, is presented. Firstly, oil remaining in solid waste (higher than 5% of waste) was recovered by the Soxhlet extraction technique, which can be useful for the soap industry. Secondly, the remaining solid was processed to yield relatively high-surface area active carbon (AC). Thirdly, the resulting carbon was employed to reversibly adsorb chromate ions from water, aiming to establish a water purification process with reusable AC. The technique used here enabled oil recovery together with the production of a clean solid, suitable for making AC. This process also has the advantage of low production cost.

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

    PubMed Central

    Weadge, Joel T.; Yip, Patrick P.; Robinson, Howard; Arnett, Krista; Tipton, Peter A.; Howell, P. Lynne

    2010-01-01

    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 Å, β = 95.7°. The crystals exhibited the symmetry of space group P21 and diffracted to a minimum d-spacing of 2.1 Å. On the basis of the Matthews coefficient (V M = 2.25 Å3 Da−1), two molecules were estimated to be present in the asymmetric unit. PMID:20445266

  9. Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC)

    PubMed Central

    Bigalke, Janna M.; Heldwein, Ekaterina E.

    2016-01-01

    The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures. This procedure may be adapted to purify and crystallize other soluble protein complexes. PMID:28042595

  10. 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.

  11. 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%.

  12. Purification of radiolabeled RNA products using denaturing gel electrophoresis

    PubMed Central

    Adachi, Hironori; Yu, Yi-Tao

    2014-01-01

    This unit discusses a basic method for purification of radiolabeled RNAs using denaturing polyacrylamide gel electrophoresis. The method consists of a number of experimental procedures, including total RNA preparation from yeast cells, isolation of a specific RNA from total yeast RNA, RNA 3' terminal labeling using nucleotide (5’[32P]pCp) addition (via ligation), denaturing (8 M urea) polyacrylamide gel electrophoresis, and RNA extraction from the gel slice. Key points for achieving good electrophoretic separation of RNA are also discussed. PMID:24510465

  13. 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.

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

    PubMed

    Gruss, Fabian; Hiller, Sebastian; Maier, Timm

    2015-01-01

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

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

    PubMed

    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

    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(1) and crystals diffracted to 1.9 A resolution at a synchrotron source. The unit-cell parameters are a = 39.75, b = 91.88, c = 48.03 A, beta = 108.37 degrees. 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.

  16. 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.

  17. Exhaust purification with on-board ammonia production

    DOEpatents

    Robel, Wade J.; Driscoll, James J.; Coleman, Gerald N.; Knox, Kevin J.

    2009-06-30

    A power source is provided for use with selective catalytic reduction systems for exhaust-gas purification. The power source includes a first cylinder group with a first air-intake passage and a first exhaust passage, and a second cylinder group with a second air-intake passage and a second exhaust passage. The second air-intake passage is fluidly isolated from the first air-intake passage. A fuel-supply device may be configured to supply fuel into the first exhaust passage, and a catalyst may be disposed downstream of the fuel-supply device to convert at least a portion of the exhaust stream in the first exhaust passage into ammonia.

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

    SciTech Connect

    Bergonzi, Celine; Schwab, Michael; Elias, Mikael

    2016-08-09

    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 bacteriumGeobacillus 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 106 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.

  19. 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.

  20. Production, Purification and Preliminary X-ray Crystallographic Studies of Adeno-Associated Virus Serotype 9

    SciTech Connect

    Mitchell, M.; Nam, H; Carter, A; McCall, A; Rence, C; Bennett, A; Gurda, B; McKenna, R; Porter, M; et. al.

    2009-01-01

    Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P32, with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetric unit capsid have been determined by molecular-replacement methods and structure determination is in progress.

  1. 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.

  2. 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.

  3. 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%.

  4. Catalytic partial oxidation coupled with membrane purification to improve resource and energy efficiency in syngas production.

    PubMed

    Iaquaniello, G; Salladini, A; Palo, E; Centi, G

    2015-02-01

    Catalytic partial oxidation coupled with membrane purification is a new process scheme to improve resource and energy efficiency in a well-established and large scale-process like syngas production. Experimentation in a semi industrial-scale unit (20 Nm(3)  h(-1) production) shows that a novel syngas production scheme based on a pre-reforming stage followed by a membrane for hydrogen separation, a catalytic partial oxidation step, and a further step of syngas purification by membrane allows the oxygen-to-carbon ratio to be decreased while maintaining levels of feed conversion. For a total feed conversion of 40 %, for example, the integrated novel architecture reduces oxygen consumption by over 50 %, with thus a corresponding improvement in resource efficiency and an improved energy efficiency and economics, these factors largely depending on the air separation stage used to produce pure oxygen.

  5. 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.

  6. Purification, crystallization and preliminary X-ray diffraction studies of C-phycocyanin and allophycocyanin from Spirulina platensis.

    PubMed

    Moreno, A; Bermejo, R; Talavera, E; Alvarez-Pez, J M; Sanz-Aparicio, J; Romero-Garrido, A

    1997-05-01

    C-phycocyanin and allophycocyanin from the green alga Spirulina platensis were isolated and crystallized by gel-acupuncture techniques. A novel two-step chromatographic procedure was used for purification. Blue hexagonal crystals were obtained by diffusing magnesium chloride into the protein solution for a week, followed by diffusion of PEG 6000 in order to complete the reduction of the solubility of the protein in the capillary tube used as a growth cell. In the case of allophycocyanin, crystals with a size of 0.4 x 0.3 x 0.3 mm were characterized by X-ray diffraction. They belong to space group P6(3)22 with unit-cell parameters a = b = 102.04, c = 131.22 A. The crystals of C-phycocyanin belong to either space group P6 or P6(3) with unit-cell constants a = b = 182.38, c = 60.87 A, alpha = beta = 90, gamma = 120 degrees. The crystals diffract beyond 2.4 and 2.5 A resolution, respectively, using a rotating anode as an X-ray source.

  7. 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 .

  8. 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.

  9. Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen Cryptococcus neoformans

    PubMed Central

    Blundell, Ross D.; Williams, Simon J.; Morrow, Carl A.; Ericsson, Daniel J.; Kobe, Bostjan; Fraser, James A.

    2013-01-01

    With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P212121 and diffracted to 2.2 Å resolution. PMID:23989157

  10. Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen Cryptococcus neoformans.

    PubMed

    Blundell, Ross D; Williams, Simon J; Morrow, Carl A; Ericsson, Daniel J; Kobe, Bostjan; Fraser, James A

    2013-09-01

    With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.2 Å resolution.

  11. Purification, crystallization and preliminary X-ray analysis of the IgV domain of human nectin-4

    PubMed Central

    Xu, Xiang; Zhang, Xiaoai; Lu, Guangwen; Cai, Yongping

    2012-01-01

    Nectin-4 belongs to a family of immunoglobulin-like cell adhesion molecules and is highly expressed in cancer cells. Recently, nectin-4 was found to be a receptor of measles virus and the IgV domain sustains strong binding to measles virus H protein. In this study, the successful expression and purification of human nectin-4 V domain (nectin-4v) is reported. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.8 Å resolution and belonged to space group P21, with unit-cell parameters a = 33.1, b = 51.7, c = 56.9 Å, β = 94.7°. Preliminary analysis of the diffraction data was also performed. PMID:22869128

  12. Soils and waste water purification from oil products using combined methods under the North conditions.

    PubMed

    Evdokimova, Galina A; Gershenkop, Alexander Sh; Mozgova, Natalia P; Myazin, Vladimir A; Fokina, Nadejda V

    2012-01-01

    Oil and gas production and transportation in Russia is increasingly moving to the north regions. Such regions are characterized by relatively low self-purification capacity of the natural environments from the contaminants due to slow character of the energy exchange and mass transfer processes. Off-shore field development in the Barents Sea and oil product transportation can result in contamination, as confirmed by the national and international practice of the developed oil and gas regions. The research aims at development of the soil bioremediation methods and industrial waste water purification contaminated by oil products in the north-western region of Russia. The dynamics of oil products carry-over have been investigated under the field model experiments in podzolic soils: gas condensate, diesel fuel and mazut from oil and the plants were selected for phyto-remediation of contaminated soils under high north latitudes. It is shown that soil purification from light hydrocarbons takes place during one vegetation period. In three months of the vegetation period the gas condensate was completely removed from the soil, diesel fuel - almost completely (more than 90%). Residual amounts of heavy hydrocarbons were traced, even 1.5 later. The following plants that were highly resistant to the oil product contamination were recommended for bioremediation: Phalaroides arundinacea, Festuca pratensis, Phleum pratense, Leymus arenarius. There has been developed and patented the combined method of treatment of waste water contaminated with hydrocarbons based on inorganic coagulants and local oil-oxidizing bacteria.

  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. Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data.

    PubMed Central

    Kordel, M; Hofmann, B; Schomburg, D; Schmid, R D

    1991-01-01

    A procedure for the purification of a very hydrophobic lipase from Pseudomonas sp. strain ATCC 21808 was elaborated by avoiding the use of long-chain detergents in view of subsequent crystallization of the enzyme. The purification procedure included chromatography on Q-Sepharose in the presence of n-octyl-beta-D-glucopyranoside, Ca2+ precipitation of fatty acids, and Octyl-Sepharose chromatography. The enzyme was purified 260-fold to a yield of 35% and a specific activity of 3,300 U/mg. The molecular weight was determined as 35,000; a polyacrylamide gel under nondenaturing conditions revealed a band at 110,000, and the isoelectric point proved to be at 4.5 to 4.6. The lipase crystallized with different salts and ethylene glycol polymers in the presence of n-octyl-beta-D-glucopyranoside and one alkyloligooxyethylene compound (CxEy) in the range from C5E2 to C8E4. The crystals diffract to a resolution of about 0.25 nm. Precession photographs revealed that they belong to space group C2 with lattice constants of a = 9.27 nm, b = 4.74 nm, c = 8.65 nm, and beta = 122.3 degrees, indicating a cell content of one molecule per asymmetric unit of the crystal. In hydrolysis of triglycerides, the lipase showed substrate specificity for saturated fatty acids from C6 to C12 and unsaturated long-chain fatty acids. Monoglycerides were hydrolyzed very slowly. The N-terminal sequence is identical to that of the lipase from Pseudomonas cepacia. Treatment with diethyl-p-nitrophenylphosphate affected the activities toward triolein and p-nitrophenylacetate to the same extent and with the same velocity. Images PMID:1856176

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

    PubMed

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

    2006-01-01

    Palatinose (isomaltulose, alpha-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 (alpha-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(1)2(1)2(1), with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 A, and diffract to 1.95 A resolution on a synchrotron-radiation source.

  16. Influence of fermentation by-products on the purification of ethanol from water using pervaporation.

    PubMed

    Chovau, S; Gaykawad, S; Straathof, A J J; Van der Bruggen, B

    2011-01-01

    Pervaporation is claimed to be a promising separation technique for the purification of ethanol from fermentation broths during bio-ethanol production. In this study, influence of fermentation by-products on the purification of ethanol from water during hydrophobic pervaporation was investigated. Sugars and salts were found to increase the membrane performance. Reason for this was a change in vapor/liquid equilibrium. 2,3-butanediol decreased the ethanol flux and selectivity factor, while glycerol exhibited no effect. This was explained by a strong sorption of butanediol into PDMS and no sorption of glycerol. Due to the presence of carboxylic acids, hydrophobicity degree of the Pervap 4060 membrane decreased, which resulted in an irreversible increase in water flux and decrease in separation performance. These observations suggested the presence of silicalite-based fillers in the membrane. When the pH was raised to a value above the dissociation constant, no changes in hydrophobicity degree and membrane performance were found.

  17. 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%.

  18. 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.

  19. 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.

  20. Expression, purification, crystallization and preliminary X-ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine

    PubMed Central

    Lashkov, Alexander A.; Gabdulkhakov, Azat G.; Prokofev, Igor I.; Seregina, Tatyana A.; Sotnichenko, Sergey E.; Lyashenko, Andrey V.; Shtil, Alexander A.; Mironov, Alexander S.; Betzel, Christian; Mikhailov, Al’bert M.

    2012-01-01

    A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of the VchUPh–thymidine complex (of dimensions ∼200–350 µm) were grown by the sitting-drop vapour-diffusion method in ∼7 d at 291 K. The crystallization solution consisted of 1.5 µl VchUPh (15 mg ml−1), 1 µl 0.1 M thymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 M MgCl2.6H2O in 0.1 M Tris–HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space group P21 (No. 4), with unit-cell parameters a = 91.80, b = 95.91, c = 91.89 Å, β = 119.96°. The Matthews coefficient was calculated as 2.18 Å3 Da−1; the corresponding solvent content was 43.74%. PMID:23143257

  1. Production and Purification of Antibodies Against Histone Modifications.

    PubMed

    Guillemette, Benoit; Hammond-Martel, Ian; Wurtele, Hugo; Verreault, Alain

    2017-01-01

    Antibodies that recognize specific histone modifications are invaluable tools to study chromatin structure and function. There are numerous commercially available antibodies that recognize a remarkable diversity of histone modifications. Unfortunately, many of them fail to work in certain applications or lack the high degree of specificity required of these reagents. The production of affinity-purified polyclonal antibodies against histone modifications demands a little effort but, in return, provides extremely valuable tools that overcome many of the concerns and limitations of commercial antibodies. We present a series of protocols and guidelines for the production and use of large amounts of polyclonal antibodies that recognize modifications of canonical histones. Our protocols can be applied to obtain antibodies that occur in histone variants and proteins other than histones. In addition, some of our protocols are compatible with the production of monoclonal or recombinant antibodies.

  2. In vitro production and purification of isochorismate using a two-enzyme cascade.

    PubMed

    Hubrich, Florian; Müller, Michael; Andexer, Jennifer N

    2014-12-10

    Combining the isochorismate synthase EntC and the chorismatase FkbO in a sequential enzyme cascade provides a useful system for the biocatalytic production and subsequent purification of isochorismate from an isochorismate/chorismate mixture. FkbO has a strict preference for chorismate - isochorismate is not accepted as a substrate - therefore the enzyme can be used to selectively hydrolyse chorismate, leading to the chiral building block 3,4-dihydroxycyclohexa-1,5-dienecarboxylate. This simplifies the final purification step, as isochorismate is much easier to separate from the chorismate hydrolysis products than from chorismate itself. The presented procedure starts with an optimised method for purifying chorismate from Escherichia coli culture supernatants, which is followed by conversion into isochorismate with the isochorismate synthase EntC, removal of the remaining chorismate by FkbO and a final purification step using an automated flash chromatography system. Isochorismate was isolated in up to 20% yield and >95% purity from chorismate, and has been characterised with respect to its degradation and suitability as a substrate in enzyme assays.

  3. Production, partial purification and characterization of xylanase using Nicotiana tabacum leaf dust as substrate.

    PubMed

    Acharya, Komal P; Shilpkar, Prateek

    2016-03-01

    Isolated Bacillus sp. was used in the present study for production of xylanase from Nicotiana tabacum leaf dust. The strain was able to give a maximum of 1.77 Uml⁻¹ xylanase activity under optimized fermentation conditions which was further increased upto 2.77 Uml⁻¹ after extraction and partial purification of enzyme. After partial purification, the enzyme was characterized and it gave the highest xylanase activity at pH 7.0, when 0.2 ml enzyme was incubated with 2.0% substrate (Nicotiana tabacum leaf dust) for 60 min at 60°C. Saccharification study of Nicotiana tabacum leaf dust with partially purified enzyme revealed that 18.4% reducing sugar was released in 20 hrs incubation, and TLC and HPTLC analysis showed that xylose and glucose sugars were obtained after hydrolysis of substrate. FTIR analysis confirmed decomposition of substrate.

  4. Exhaust purification with on-board ammonia production

    DOEpatents

    Robel, Wade J.; Driscoll, James Joshua; Coleman, Gerald N.

    2008-05-13

    A system of ammonia production for a selective catalytic reduction system is provided. The system includes producing an exhaust gas stream within a cylinder group, wherein the first exhaust gas stream includes NOx. The exhaust gas stream may be supplied to an exhaust passage and cooled to a predetermined temperature range, and at least a portion of the NOx within the exhaust gas stream may be converted into ammonia.

  5. Exhaust purification with on-board ammonia production

    DOEpatents

    Robel, Wade J.; Driscoll, James Joshua; Coleman, Gerald N.

    2010-10-12

    A method of ammonia production for a selective catalytic reduction system is provided. The method includes producing an exhaust gas stream within a cylinder group, wherein the first exhaust gas stream includes NOx. The exhaust gas stream may be supplied to an exhaust passage and cooled to a predetermined temperature range, and at least a portion of the NOx within the exhaust gas stream my be converted into ammonia.

  6. General introduction: recombinant protein production and purification of insoluble proteins.

    PubMed

    Ferrer-Miralles, Neus; Saccardo, Paolo; Corchero, José Luis; Xu, Zhikun; García-Fruitós, Elena

    2015-01-01

    Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and the most appropriate growth conditions to minimize the formation of insoluble proteins should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

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

    PubMed Central

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

    2014-01-01

    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 Å3 Da−1 and a solvent content of 73.23%. PMID:24598926

  8. 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

  9. 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.

  10. Improved purification, crystallization and crystallographic study of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77.

    PubMed

    Muhd Noor, Noor Dina; Nishikawa, Koji; Nishihara, Hirofumi; Yoon, Ki Seok; Ogo, Seiji; Higuchi, Yoshiki

    2016-01-01

    The purification procedure of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77 was improved by applying treatment with trypsin before chromatography. Purified protein samples both with and without trypsin treatment were successfully crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol as a precipitant. Both crystals belonged to space group P21, with unit-cell parameters a = 63.90, b = 118.89, c = 96.70 Å, β = 100.61° for the protein subjected to trypsin treatment and a = 65.38, b = 121.45, c = 98.63 Å, β = 102.29° for the sample not treated with trypsin. The crystal obtained from the trypsin-treated protein diffracted to 1.60 Å resolution, which is considerably better than the 2.00 Å resolution obtained without trypsin treatment. The [NiFe]-hydrogenase from Citrobacter sp. S-77 retained catalytic activity with some amount of O2, indicating that it has clear O2 tolerance.

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

    PubMed

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

    2012-11-01

    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 P2(1)2(1)2(1), 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.

  12. 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.

  13. 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.

  14. 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.

  15. Production, purification, and characterization of exoglucanase by Aspergillus fumigatus.

    PubMed

    Mahmood, Raja Tahir; Asad, Muhammad Javaid; Mehboob, Nazia; Mushtaq, Maria; Gulfraz, Muhammad; Asgher, Muhammad; Minhas, Nasir M; Hadri, Saqib Hussain

    2013-06-01

    Fungi are considered good producers of industrially valuable enzymes with higher enzymatic activities. Among these cellulases are group of extracellular enzymes commonly employed in many industries for the hydrolysis of cellulolytic material. Aspergillus fumigatus produced exoglucanase having high enzymatic activity (83 U/gds) during the solid-state fermentation of wheat straw under optimum physical and nutritional conditions. Maximum production was obtained after 72 h of fermentation, at 55 °C temperature, pH 5.5, 80 % moisture level, and 2 mL fungal inoculum. Production was further increased by the addition of fructose (0.3 %) as additional carbon source, peptone (0.4 %) as nitrogen source, Tween-80 (0.3 %) as surfactant, and ammonium sulfate (0.2 %) in media. Exoglucanase was 2.30-folds purified by adding 40 % ammonium sulfate with volumetric activity 95.4 U/gds and specific activity 14.74 U/mg. Further, it was 5.18-folds purified by gel filtration chromatography with volumetric activity 115.2 U/gds and specific activity 33.10 U/mg. Purified exoglucanase has maximum activity at 55 °C and pH 4.8 using 1 % Avicel aqueous solution as substrate. The K(m) and V(max) were 4.34 mM and 7.29 μM/min, respectively. Calcium, magnesium, and zinc ions have positive effect on exoglucanase activity.

  16. Laccase: Microbial Sources, Production, Purification, and Potential Biotechnological Applications

    PubMed Central

    Shraddha; Shekher, Ravi; Sehgal, Simran; Kamthania, Mohit; Kumar, Ajay

    2011-01-01

    Laccase belongs to the blue multicopper oxidases and participates in cross-linking of monomers, degradation of polymers, and ring cleavage of aromatic compounds. It is widely distributed in higher plants and fungi. It is present in Ascomycetes, Deuteromycetes and Basidiomycetes and abundant in lignin-degrading white-rot fungi. It is also used in the synthesis of organic substance, where typical substrates are amines and phenols, the reaction products are dimers and oligomers derived from the coupling of reactive radical intermediates. In the recent years, these enzymes have gained application in the field of textile, pulp and paper, and food industry. Recently, it is also used in the design of biosensors, biofuel cells, as a medical diagnostics tool and bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases have received attention of researchers in the last few decades due to their ability to oxidize both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. It has been identified as the principal enzyme associated with cuticular hardening in insects. Two main forms have been found: laccase-1 and laccase-2. This paper reviews the occurrence, mode of action, general properties, production, applications, and immobilization of laccases within different industrial fields. PMID:21755038

  17. A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification.

    PubMed

    Hu, Jian; Qin, Huajun; Gao, Fei Philip; Cross, Timothy A

    2011-11-01

    Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.

  18. 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.

  19. 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.

  20. 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.

  1. Purification, crystallization and preliminary X-ray studies of two isoforms of Rubisco from Alcaligenes eutrophus.

    PubMed

    Hansen, S; Hough, E; Andersen, K

    1999-01-01

    Two different isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Alcaligenes eutrophus have been purified and crystallized. Both isoforms crystallize in space group P43212. Crystals of isoform I (unit-cell dimensions a = 112.0 and c = 402.7 A) diffract to 2.7 A, whereas isoform II (unit-cell dimensions a = 111.8 and c = 400.0 A) presently diffract to 3.2 A, using synchrotron radiation in both cases.

  2. ProteinTracker: an application for managing protein production and purification

    PubMed Central

    2012-01-01

    Background Laboratories that produce protein reagents for research and development face the challenge of deciding whether to track batch-related data using simple file based storage mechanisms (e.g. spreadsheets and notebooks), or commit the time and effort to install, configure and maintain a more complex laboratory information management system (LIMS). Managing reagent data stored in files is challenging because files are often copied, moved, and reformatted. Furthermore, there is no simple way to query the data if/when questions arise. Commercial LIMS often include additional modules that may be paid for but not actually used, and often require software expertise to truly customize them for a given environment. Findings This web-application allows small to medium-sized protein production groups to track data related to plasmid DNA, conditioned media samples (supes), cell lines used for expression, and purified protein information, including method of purification and quality control results. In addition, a request system was added that includes a means of prioritizing requests to help manage the high demand of protein production resources at most organizations. ProteinTracker makes extensive use of existing open-source libraries and is designed to track essential data related to the production and purification of proteins. Conclusions ProteinTracker is an open-source web-based application that provides organizations with the ability to track key data involved in the production and purification of proteins and may be modified to meet the specific needs of an organization. The source code and database setup script can be downloaded from http://sourceforge.net/projects/proteintracker. This site also contains installation instructions and a user guide. A demonstration version of the application can be viewed at http://www.proteintracker.org. PMID:22574679

  3. The role of purification in the crystallization of proteins and nucleic acids

    NASA Astrophysics Data System (ADS)

    Giegé, R.; Dock, A. C.; Kern, D.; Lorber, B.; Thierry, J. C.; Moras, D.

    1986-08-01

    In structural biology, the crystallization of the macromolecules often represents the most challenging step. Beside classical factors which determine the solubility of macromolecules, purity of compounds is another major parameter governing crystal growth. With aminoacyl-tRNA synthetases and transfer ribonucleic acids as examples, it will be shown that molecules to be crystallized not only have to be pure in terms of contaminating molecules, but also in terms of sequence integrity and conformational homogeneity. A chromatographic method based on salting-out of proteins or nucleic acids on Sepharose 4B gels and back-solubilization with inverse salt gradients will be discussed in the light of crystal growth experiments.

  4. Thermal-destruction products of coal in the blast-furnace gas-purification system

    SciTech Connect

    A.M. Amdur; M.V. Shibanova; E.V. Ental'tsev

    2008-10-15

    The lean, poorly clinkering coal and anthracite used to replace coke in blast furnaces has a considerable content of volatile components (low-molecular thermaldestruction products), which enter the water and sludge of the blast-furnace gas-purification system as petroleum products. Therefore, it is important to study the influence of coal on the petroleum-product content in the water and sludge within this system. The liberation of primary thermal-destruction products is investigated for anthracite with around 4 wt % volatiles, using a STA 449C Jupiter thermoanalyzer equipped with a QMC 230 mass spectrometer. The thermoanalyzer determines small changes in mass and thermal effects with high accuracy (weighing accuracy 10{sup -8} g; error in measuring thermal effects 1 mV). This permits experiments with single layers of coal particles, eliminating secondary reactions of its thermal-destruction products.

  5. Purification, crystallization and preliminary X-ray structure analysis of the banana lectin from Musa paradisiaca.

    PubMed

    Singh, D D; Saikrishnan, K; Kumar, Prashant; Dauter, Z; Sekar, K; Surolia, A; Vijayan, M

    2004-11-01

    The banana lectin from Musa paradisiaca, MW 29.4 kDa, has been isolated, purified and crystallized. The trigonal crystals contain one dimeric molecule in the asymmetric unit. The structure has been solved using molecular replacement to a resolution of 3 A. The structure of the subunit is similar to that of jacalin-like lectins.

  6. 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.

  7. Protein products obtained by site-preferred partial crosslinking in protein crystals and "liberated" by redissolution.

    PubMed

    Buch, Michal; Wine, Yariv; Dror, Yael; Rosenheck, Sonia; Lebendiker, Mario; Giordano, Rita; Leal, Ricardo M F; Popov, Alexander N; Freeman, Amihay; Frolow, Felix

    2014-07-01

    The use of protein crystals as a source of nanoscale biotemplates has attracted growing interest in recent years owing to their inherent internal order. As these crystals are vulnerable to environmental changes, potential applications require their stabilization by chemical crosslinking. We have previously shown that such intermolecular chemical crosslinking reactions occurring within protein crystals are not random events, but start at preferred crosslinking sites imposed by the alignment of protein molecules and their packing within the crystalline lattice. Here we propose a new working hypothesis and demonstrate its feasibility in enabling us to extricate homogeneous populations of single protein molecules that display chemical point mutations or of dimers that show homogeneous chemical crosslinking, and that have the potential for isolation of higher structures. Characterization of the crosslinking mechanism and its end products opens the way to the potential retrieval of such specific modified/intermolecular crosslinked products simply by effecting partial crosslinking at identified preferred sites, followed by time-controlled arrest of the crosslinking reaction and dissolution of the crystals by medium exchange complemented by chromatographic purification.

  8. Purification, crystallization and preliminary characterization of a putative LmbE-like deacetylase from Bacillus cereus

    SciTech Connect

    Fadouloglou, Vasiliki E.; Kotsifaki, Dina; Gazi, Anastasia D.; Fellas, Georgios; Meramveliotaki, Chrysi; Deli, Alexandra; Psylinakis, Emmanuel; Bouriotis, Vassilis; Kokkinidis, Michael

    2006-03-01

    The BC1534 protein from B. cereus was purified and crystallized and a native X-ray diffraction data set was collected to 2.5 Å using synchrotron radiation. The Bacillus cereus BC1534 protein, a putative deacetylase from the LmbE family, has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Crystals of the 26 kDa protein grown from MPD and acetate buffer belong to space group R32, with unit-cell parameters a = b = 76.7, c = 410.5 Å (in the hexagonal setting). A complete native data set was collected to a resolution of 2.5 Å from a single cryoprotected crystal using synchrotron radiation. As BC1534 shows significant sequence homology with an LmbE-like protein of known structure from Thermus thermophilus, molecular replacement will be used for crystal structure determination.

  9. 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.

  10. 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°.

  11. [Adeno-associated viral vectors: methods for production and purification for gene therapy applications].

    PubMed

    Mena-Enriquez, Mayra; Flores-Contreras, Lucia; Armendáriz-Borunda, Juan

    2012-01-01

    Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials.

  12. Solvent-resistant nanofiltration for product purification and catalyst recovery in click chemistry reactions.

    PubMed

    Cano-Odena, Angels; Vandezande, Pieter; Fournier, David; Van Camp, Wim; Du Prez, Filip E; Vankelecom, Ivo F J

    2010-01-18

    The quickly developing field of "click" chemistry would undoubtedly benefit from the availability of an easy and efficient technology for product purification to reduce the potential health risks associated with the presence of copper in the final product. Therefore, solvent-resistant nanofiltration (SRNF) membranes have been developed to selectively separate "clicked" polymers from the copper catalyst and solvent. By using these solvent-stable cross-linked polyimide membranes in diafiltration, up to 98 % of the initially present copper could be removed through the membrane together with the DMF solvent, the polymer product being almost completely retained. This paper also presents the first SRNF application in which the catalyst permeates through the membrane and the reaction product is retained.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. Optimization of Growth Conditions for Purification and Production of L-Asparaginase by Spirulina maxima.

    PubMed

    Abd El Baky, Hanaa H; El Baroty, Gamal S

    2016-01-01

    L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA from Spirulina maxima (SM) were tested. SM cultures grown in Zarrouk medium containing different N2 (in NaNO3 form) concentrations (1.25, 2.50, and 5.0 g/L) for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2 concentration. The cultures of SM at large scales (300 L medium, 5 g/L N2) showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2 cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production in S. maxima.

  18. Optimization of Growth Conditions for Purification and Production of L-Asparaginase by Spirulina maxima

    PubMed Central

    El Baroty, Gamal S.

    2016-01-01

    L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA from Spirulina maxima (SM) were tested. SM cultures grown in Zarrouk medium containing different N2 (in NaNO3 form) concentrations (1.25, 2.50, and 5.0 g/L) for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2 concentration. The cultures of SM at large scales (300 L medium, 5 g/L N2) showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2 cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production in S. maxima. PMID:27525017

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

    SciTech Connect

    Balasubramanian, Anuradha; Ponnuraj, Karthe

    2008-07-01

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

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

    NASA Astrophysics Data System (ADS)

    Sarikaya, Elif; Mikami, Bunzo

    2001-11-01

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

  1. Purification, crystallization and preliminary X-ray crystallographic analysis of chitinase from Bacillus cereus NCTU2

    SciTech Connect

    Kuo, Chueh-Yuan; Wu, Yue-Jin; Hsieh, Yin-Cheng; Guan, Hong-Hsiang; Tsai, Huei-Ju; Lin, Yi-Hung; Huang, Yen-Chieh; Liu, Ming-Yih; Li, Yaw-Kuen; Chen, Chun-Jung

    2006-09-01

    The crystallization of B. cereus chitinase is reported. Chitinases (EC 3.2.1.14) are found in a broad range of organisms, including bacteria, fungi and higher plants, and play different roles depending on their origin. A chitinase from Bacillus cereus NCTU2 (ChiNCTU2) capable of hydrolyzing chitin as a carbon and nitrogen nutrient has been identified as a member of the family 18 glycoside hydrolases. ChiNCTU2 of molecular weight 36 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of chitinase crystals at 1.10 Å resolution, the crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 Å, β = 99.31°. Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.

  2. 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.

  3. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

    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. P21 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.

  5. Purification, crystallization and preliminary characterization of a putative LmbE-like deacetylase from Bacillus cereus

    PubMed Central

    Fadouloglou, Vasiliki E.; Kotsifaki, Dina; Gazi, Anastasia D.; Fellas, Georgios; Meramveliotaki, Chrysi; Deli, Alexandra; Psylinakis, Emmanuel; Bouriotis, Vassilis; Kokkinidis, Michael

    2006-01-01

    The Bacillus cereus BC1534 protein, a putative deacetylase from the LmbE family, has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Crystals of the 26 kDa protein grown from MPD and acetate buffer belong to space group R32, with unit-cell parameters a = b = 76.7, c = 410.5 Å (in the hexagonal setting). A complete native data set was collected to a resolution of 2.5 Å from a single cryoprotected crystal using synchrotron radiation. As BC1534 shows significant sequence homology with an LmbE-like protein of known structure from Thermus thermophilus, molecular replacement will be used for crystal structure determination. PMID:16511317

  6. 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.

  7. 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.

  8. 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.

  9. A Cost-Effective ELP-Intein Coupling System for Recombinant Protein Purification from Plant Production Platform

    PubMed Central

    Tian, Li; Sun, Samuel S. M.

    2011-01-01

    Background Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor. Methodology/Principal Findings To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL) with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP) fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV). The in vivo uncleaved EiP protein was accumulated up to 2–4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein. Conclusion/Significance This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry. PMID:21918684

  10. Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidus d-arabinose isomerase

    PubMed Central

    Takeda, Kosei; Yoshida, Hiromi; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2008-01-01

    d-Arabinose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. Bacillus pallidus d-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of d-arabinose, l-fucose, l-xylose, l-galactose and d-­altrose. Recombinant B. pallidus d-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 Å. Diffraction data were collected to 2.3 Å resolution. PMID:18931442

  11. Proanthocyanidin A2 purification and levels found in American cranberry (Vaccinium macrocarpon Ait.) products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, five common proanthocyanidin purification techniques were evaluated prior to phloroglucinolysis, followed by HPLC analysis. An optimized purification method was then used to identify and quantify the proanthocyanidins (extension and terminal units of epigallocatechin, catechin, epicat...

  12. Production, purification and characterization of laccase from Pleurotus ostreatus grown on tomato pomace.

    PubMed

    Freixo, Maria do Rosário; Karmali, Amin; Arteiro, José Maria

    2012-01-01

    A strain of Pleurotus ostreatus was grown in tomato pomace as sole carbon source for production of laccase. The culture of P. ostreatus revealed a peak of laccase activity (147 U/L of fermentation broth) on the 4th day of culture with a specific activity of 2.8 U/mg protein. Differential chromatographic behaviour of laccase was investigated on affinity chromatographic matrices containing either urea, acetamide, ethanolamine or IDA as affinity ligands. Laccase exhibited retention on such affinity matrices and it was purified on a Sepharose 6B-BDGE-urea column with final enzyme recoveries of about 60%, specific activity of 6.0 and 18.0 U/mg protein and purification factors in the range of 14-46. It was also possible to demonstrate that metal-free laccase did not adsorb to Sepharose 6B-BDGE-urea column which suggests that adsorption of native laccase on this affinity matrix was apparently due to the specific interaction of carbonyl groups available on the matrix with the active site Cu (II) ions of laccase. The kinetic parameters (V(max), K(m), K(cat), and K(cat)/K(m)) of the purified enzyme for several substrates were determined as well as laccase stability and optimum pH and temperature of enzyme activity. This is the first report describing the production of laccase from P. ostreatus grown on tomato pomace and purification of this enzyme based on affinity matrix containing urea as affinity ligand.

  13. Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

    PubMed

    Boksha, I S; Lavrova, N V; Grishin, A V; Demidenko, A V; Lyashchuk, A M; Galushkina, Z M; Ovchinnikov, R S; Umyarov, A M; Avetisian, L R; Chernukha, M Iu; Shaginian, I A; Lunin, V G; Karyagina, A S

    2016-05-01

    Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

  14. 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.

  15. 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.

  16. Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from Pyrococcus horikoshii

    SciTech Connect

    Inagaki, Eiji; Sakamoto, Keiko; Obayashi, Naomi; Terada, Takaho; Shirouzu, Mikako; Bessho, Yoshitaka; Kuroishi, Chizu; Kuramitsu, Seiki; Shinkai, Akeo; Yokoyama, Shigeyuki

    2006-02-01

    Galactokinase from P. horikoshii has been crystallized in both the apo form and as a ternary complex with α-d-galactose and an ATP analogue. The crystals were characterized by X-ray diffraction. The kinetic parameters of the enzyme were determined. Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of α-d-galactose to α-d-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with α-d-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 Å resolution for the apo form and to 1.7 Å for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 Å, β = 109.8°. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

  17. 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.

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

    PubMed Central

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

    2012-01-01

    The periplasmic alginate lyase AlgL is essential for the synthesis and export of the exopolysaccharide alginate in Pseudomonas sp. and also plays a role in its depolymerization. P. aeruginosa PAO1 AlgL has been overexpressed and purified and diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as thin plates, with unit-cell parameters a = 56.4, b = 59.6, c = 102.1 Å, α = β = γ = 90°. The AlgL crystals exhibited the symmetry of space group P212121 and diffracted to a minimum d-­spacing of 1.64 Å. Based on the Matthews coefficient (V M = 2.20 Å3 Da−1), one molecule is estimated to be present in the asymmetric unit. PMID:22691793

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

    PubMed Central

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

    2008-01-01

    Inositol dehydrogenase (IDH) is an enzyme that catalyses the NAD+-dependent oxidation of myo-inositol to scyllo-inosose. The enzyme has been purified to homogeneity by means of Ni2+-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. PMID:18259059

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Expression, purification, crystallization and structure of human adipocyte lipid-binding protein (aP2)

    SciTech Connect

    Marr, Eric; Tardie, Mark; Carty, Maynard; Brown Phillips, Tracy; Wang, Ing-Kae; Soeller, Walt; Qiu, Xiayang Karam, George

    2006-11-01

    The crystal structure of human adipocyte lipid-binding protein (aP2) with a bound palmitate is reported at 1.5 Å resolution. Human adipocyte lipid-binding protein (aP2) belongs to a family of intracellular lipid-binding proteins involved in the transport and storage of lipids. Here, the crystal structure of human aP2 with a bound palmitate is described at 1.5 Å resolution. Unlike the known crystal structure of murine aP2 in complex with palmitate, this structure shows that the fatty acid is in a folded conformation and that the loop containing Phe57 acts as a lid to regulate ligand binding by excluding solvent exposure to the central binding cavity.

  5. 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.

  6. 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.

  7. 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.

  8. Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracis

    SciTech Connect

    Misra, Gauri; Aggarwal, Anita; Mittal, Sonia; Singh, Yogendra; Ramachandran, Ravishankar

    2007-12-01

    Nucleoside diphosphate kinase from B. anthracis has been crystallized. Preliminary crystallographic analysis shows that there is one monomer in the asymmetric unit of the crystal. Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2 µl 10 mg ml{sup −1} protein in 50 mM Tris–HCl pH 8.0, 50 mM NaCl, 5 mM EDTA equilibrated against 500 µl reservoir solution consisting of 2.25 M ammonium formate and 0.1 M HEPES buffer pH 7.25. Diffraction data extending to 2.0 Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3 Å. The crystals are hexagonal in shape and belong to space group P6{sub 3}22. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V{sub M}) of 2.1 Å{sup 3} Da{sup −1} and a solvent content of about 36.9%.

  9. 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.

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

    PubMed Central

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

    2009-01-01

    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 ∼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 P41, with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 Å. PMID:19342775

  11. 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

  12. Cloning, overproduction, purification, crystallization and preliminary X-ray diffraction analysis of yeast glutaredoxin Grx5.

    PubMed

    Wang, Yi; He, Yong Xing; Yu, Jiang; Zhou, Cong Zhao

    2009-06-01

    Grx5 from the yeast Saccharomyces cerevisiae is a monothiol glutaredoxin that is involved in iron-sulfur cluster biogenesis. Here, yeast Grx5 was cloned and overproduced in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Diffraction data for Grx5 were collected to 1.67 A resolution. The crystal of Grx5 belonged to space group R3, with unit-cell parameters a = b = 85.12, c = 48.95 A, alpha = beta = 90.00, gamma = 120.00 degrees .

  13. Production and purification of recombinant human hepcidin-25 with authentic N and C-termini.

    PubMed

    Janakiraman, Vignesh Narasimhan; Cabanne, Charlotte; Dieryck, Wilfrid; Hocquellet, Agnès; Joucla, Gilles; Le Senechal, Caroline; Chaignepain, Stephane; Costaglioli, Patricia; Santarelli, Xavier; Garbay, Bertrand; Noubhani, Abdelmajid

    2015-02-10

    Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long-sought hormone that regulates iron homeostasis in mammals. Recombinant human Hepcidin-25 (Hepc25) was expressed in Pichia pastoris using a modified version of the pPICZαA vector. Hepc25 was then purified by a simple two-step chromatographic process to obtain 1.9 mg of soluble recombinant human Hepc25 per liter of culture at 96% purity. The sequence of Hepc25 and the presence of four disulfide bridges were confirmed by mass spectrometry analyses, and the recombinant Hepc25 exhibited antibacterial activity. This protocol of production and purification is the first step toward the production of human Hepc25 at a greater scale.

  14. Production and purification of amylolytic enzymes for saccharification of microalgal biomass.

    PubMed

    Rodrigues, Éllen Francine; Ficanha, Aline Matuella Moreira; Dallago, Rogério Marcos; Treichel, Helen; Reinehr, Christian Oliveira; Machado, Tainara Paula; Nunes, Greice Borges; Colla, Luciane Maria

    2017-02-01

    The aim of this study was the production of amylolytic enzymes by solid state or submerged fermentations (SSF or SF, respectively), followed by purification using chemical process or microfiltration and immobilization of purified enzymes in a polyurethane support. The free and immobilized enzymes obtained were used to evaluate enzymatic hydrolysis of the polysaccharides of Spirulina. Microfiltration of the crude extracts resulted in an increase in their specific activity and thermal stability at 40°C and 50°C for 24h, as compared to extracts obtained by SSF and SF. Immobilization of polyurethane purified enzyme produced yields of 332% and 205% for the enzymes obtained by SF and SSF, respectively. Free or immobilized enzymes favor the generation of fermentable sugar, being the application of the purified and immobilized enzymes in the hydrolysis of microalgal polysaccharides considered a promising alternative towards development of the bioethanol production process from microalgal biomass.

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

    SciTech Connect

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

    2006-07-01

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

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

    PubMed Central

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

    2006-01-01

    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 M is calculated to be 2.4 Å3 Da−1, assuming there to be 12 protein molecules in the asymmetric unit. PMID:17142914

  17. 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.

  18. 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.

  19. RESEARCH IN PURIFICATION AND SINGLE CRYSTAL GROWTH OF II-VI COMPOUNDS.

    DTIC Science & Technology

    on the zoning of a doped cadmium test bar is presented. The syntheses of high purity cadmium sulfide, cadmium selenide , zinc sulfide, and zinc...ray characterization of this material are presented. Crystal growth of cadmium sulfide, zinc sulfide, cadmium selenide , zinc selenide, and mixed

  20. 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.

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

    PubMed Central

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

    2007-01-01

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

  2. Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

    PubMed

    Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

    2011-05-01

    For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

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

    PubMed Central

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

    2007-01-01

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

  4. Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents.

    PubMed

    Turner, Paul R; Bourne, Katie; Garama, Daniel; Carne, Alan; Abraham, Wickliffe C; Tate, Warren P

    2007-08-15

    The secreted fragment of the amyloid precursor protein (sAPPalpha) generated following cleavage by alpha-secretase is an important mediator of cell function and is both neurotrophic and neuroprotective. HEK 293T cells have been stably integrated with a fragment of the APP gene to produce and secrete either sAPPalpha, or the alternative cleavage product sAPPbeta. Heparin binding domains on the proteins have been utilised to develop a one-step fast-performance-liquid-chromatography (FPLC) purification of sAPPs from the conditioned media. Immunoblotting analyses with a sAPP specific antibody coupled with highly sensitive silver staining techniques have validated the expression and purification strategy. Functional activity of the purified fragments was demonstrated by their ability to protect COS-7 and SH-SY5Y (neuroblastoma) cells against the adverse effects of glucose deprivation in a cell viability assay. The purified sAPPs also activated the NFkappaB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPalpha the more potent activator as expected. The simple protocol to produce these mammalian expressed proteins will facilitate their use as potential neuropharmacological reagents in the elucidation of biochemical pathways modulated by sAPPs, and in the study of Alzheimer's disease mechanisms in general.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Expression, purification, crystallization, and preliminary X-Ray analysis of the human UDP-glucose dehydrogenase.

    PubMed

    Huh, Jae Wan; Robinson, Robert Charles; Lee, Han Sam; Lee, Jae Il; Heo, Yong Seok; Kim, Hyun Tae; Lee, Hyun Ju; Cho, Sung Woo; Choe, Han

    2006-01-01

    UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis of UDP-glucuronic acid from UDP-glucose resulting in the formation of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers. Here, human UGDH (hUGDH) was purified and crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data were collected to a resolution of 2.8 A. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 A, and alpha = beta = gamma = 90.0 degrees. Based on preliminary analysis of the diffraction data, we propose that the biological unit of hUGDH is a tetramer.

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

    PubMed Central

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

    2008-01-01

    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 P21, with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution. PMID:18259070

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

    PubMed

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

    2012-11-01

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

  14. 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}.

  15. 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

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

    SciTech Connect

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

    2007-11-01

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

  17. Purification, crystallization and data collection of methicillin-resistant Staphylococcus aureus Sar2676, a pantothenate synthetase

    PubMed Central

    Seetharamappa, Jaldappagari; Oke, Muse; Liu, Huanting; McMahon, Stephen A.; Johnson, Kenneth A.; Carter, Lester; Dorward, Mark; Zawadzki, Michal; Overton, Ian M.; van Niekirk, C. A. Johannes; Graham, Shirley; Botting, Catherine H.; Taylor, Garry L.; White, Malcolm F.; Barton, Geoffrey J.; Coote, Peter J.; Naismith, James H.

    2007-01-01

    Sar2676, a pantothenate synthetase with a molecular weight of 31 419 Da from methicillin-resistant Staphylococcus aureus, has been expressed, purified and crystallized at 293 K. The protein crystallizes in a primitive triclinic lattice, with unit-cell parameters a = 45.3, b = 60.5, c = 117.6 Å, α = 87.2, β = 81.2, γ = 68.4°. A complete data set has been collected to 2.3 Å resolution at the ESRF. Consideration of the likely solvent content suggested the asymmetric unit to contain four molecules. This has been confirmed by molecular-replacement phasing calculations, which give a solution with four monomers using a monomer of pantothenate synthetase from Escherichia coli (PDB code 1iho), which is 41% identical to Sar2676, as a search model. PMID:17554169

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

    PubMed Central

    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

    2006-01-01

    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 C2221 (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 trypano­somatids. PMID:16880563

  19. 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.

  20. 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.

  1. A cell-free expression and purification process for rapid production of protein biologics.

    PubMed

    Sullivan, Challise J; Pendleton, Erik D; Sasmor, Henri H; Hicks, William L; Farnum, John B; Muto, Machiko; Amendt, Eric M; Schoborg, Jennifer A; Martin, Rey W; Clark, Lauren G; Anderson, Mark J; Choudhury, Alaksh; Fior, Raffaella; Lo, Yu-Hwa; Griffey, Richard H; Chappell, Stephen A; Jewett, Michael C; Mauro, Vincent P; Dresios, John

    2016-02-01

    Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.

  2. 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.

  3. 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

  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, 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

  6. 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

  7. Purification, crystallization and preliminary crystallographic analysis of mouse myo-inositol oxygenase

    SciTech Connect

    Brown, Peter M. Caradoc-Davies, Tom T.; Dickson, James M.; Cooper, Garth J. S.; Loomes, Kerry M.; Baker, Edward N.

    2006-08-01

    Mouse myo-inositol oxygenase, a key enzyme involved in inositol catabolism, has been expressed, purified and crystallized in a form suitable for structure determination by X-ray crystallography. Myo-inositol oxygenase (MIOX) catalyzes the novel oxidative cleavage of myo-inositol (MI) and its epimer d-chiro inositol (DCI) to d-glucuronate. MIOX utilizes an Fe{sup II}/Fe{sup III} binuclear iron centre for the dioxygen-dependent cleavage of the C1—C6 bond in MI. Despite its key role in inositol metabolism, the structural basis of its unique four-electron oxidation mechanism and its substrate specificity remain unknown. In order to answer these questions and to facilitate the use of this key enzyme for the development of new therapeutic strategies for diabetes, the mouse enzyme has been cloned, expressed in Escherichia coli, purified and crystallized from 4.4 M sodium formate. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.87, b = 77.26, c = 84.84 Å, and diffract to 2.8 Å resolution.

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

    SciTech Connect

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

    2007-04-01

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

  9. 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.

  10. Production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans 191

    PubMed Central

    Fleuri, Luciana F.; Kawaguti, Haroldo Y.; Sato, Hélia H.

    2009-01-01

    This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25°C and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25°C and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts. PMID:24031407

  11. 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.

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

    PubMed

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

    2007-11-01

    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.

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

    PubMed

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

    2015-01-01

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

  14. 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.

  15. Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

    PubMed Central

    Coleman, Jonathan A.; Green, Evan M.; Gouaux, Eric

    2016-01-01

    The serotonin transporter is a sodium and chloride-coupled transporter that "pumps" extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 Å resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins. PMID:27929454

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

    PubMed Central

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

    2007-01-01

    The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His6-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 Å resolution. Crystals belonged to the tetragonal space group P43, with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å3 Da−1, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism. PMID:17401200

  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, Assay, and Crystal Structure of Perdeuterated Human Arginase I

    SciTech Connect

    Di Costanzo,L.; Moulin, M.; Haertlein, M.; Meilleur, F.; Christianson, D.

    2007-01-01

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

  19. 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.

  20. Production, Purification, and Characterization of a Major Penicillium glabrum Xylanase Using Brewer's Spent Grain as Substrate

    PubMed Central

    Beitel, Susan Michelz; Fortkamp, Diana; Terrasan, César Rafael Fanchini; de Almeida, Alex Fernando

    2013-01-01

    In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. PMID:23762855

  1. Production, purification, and characterization of a major Penicillium glabrum xylanase using Brewer's spent grain as substrate.

    PubMed

    Knob, Adriana; Beitel, Susan Michelz; Fortkamp, Diana; Terrasan, César Rafael Fanchini; de Almeida, Alex Fernando

    2013-01-01

    In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn(2+) and the reducing agents β -mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg(2+), Zn(2+), and Cu(2+) as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.

  2. A panorama of bacterial inulinases: Production, purification, characterization and industrial applications.

    PubMed

    Singh, Ram Sarup; Chauhan, Kanika; Kennedy, John F

    2017-03-01

    Inulinases are important hydrolysing enzymes which specifically act on β-2, 1 linkages of inulin to produce fructose or fructooligosaccharides. Fungi, yeasts and bacteria are the potent microbial sources of inulinases. The data on bacterial inulinases is scarce as compared to other microbial sources. Inulinases yield from bacteria is very less as compared to fungal and yeast sources of inulinases. Submerged fermentation (SmF) is the method of choice for the production of inulinases from bacterial sources. Moreover, inulin is a potent substrate for the production of inulinases in SmF. Many bacterial inulinases have been reported to display magnificent environment abiding features and variability in their biophysical and biochemical properties. These properties have attracted intention of many researchers towards exploring adverse ecological niches for more distinctive inulinase producing bacterial strains. Inulinases are substantially important in current biotechnological era due to their numerous industrial applications. High fructose syrup and fructooligosaccharides are two major industrial applications of inulinases. Additionally, there are many reports on the production of various metabolites like citric acid, lactic acid, ethanol, biofuels, butanediol etc. using mixed cultures of inulinase producing organisms with other microorganisms. The present review mainly envisages inulinase producing bacterial sources, inulinase production, purification, characterization and their applications.

  3. A comprehensive classification of solvent systems used for natural product purifications in countercurrent and centrifugal partition chromatography.

    PubMed

    Skalicka-Woźniak, Krystyna; Garrard, Ian

    2015-11-01

    Using both library paper copies and modern electronic copies, every known, published, English-language journal paper that employs either countercurrent or centrifugal partition chromatography solvent systems for natural product purifications has been studied and the solvent systems classified in a comprehensive database. Papers were studied from the earliest found examples containing natural product separations in 1984 until the end of 2014. In total, 2594 solvent systems have been classified, of which 272 are gradient systems. To observe any trends or patterns in the data, the natural product solutes were divided into 21 classes and the solvent systems into 7 different types. The complete database, sorted according to natural product class, is available for download to assist separation scientists in future liquid-liquid chromatography purifications.

  4. Phosphoprotein affinity purification identifies proteins involved in S-adenosyl-L-methionine-induced enhancement of antibiotic production in Streptomyces coelicolor.

    PubMed

    Meng, Lingzhu; Yang, Seung Hwan; Palaniyandi, Sasikumar Arunachalam; Lee, Sung-Kwon; Lee, In-Ae; Kim, Tae-Jong; Suh, Joo-Won

    2011-01-01

    Streptomycetes are the major natural source of clinical antibiotics. The enhanced secondary metabolite production of many streptomycetes by S-adenosylmethionine (SAM) in previous studies suggested the existence of a common SAM regulatory effect. We screened nine proteins using the phosphoprotein purification column from Streptomyces coelicolor. Among them, genes (SCO5477, SCO5113, SCO4647, SCO4885 and SCO1793) for five proteins were disrupted by insertion mutation. The undecylprodigiosin and actinorhodin productions were changed in all mutations. The SAM-induced enhancement of actinorhodin production was abolished by all mutations except SCO4885 mutation, which reduced the production of actinorhodin and undecylprodigiosin with SAM treatment. This study demonstrates that phosphoprotein affinity purification can be used as a screening method to identify the proteins involved SAM signaling.

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

    PubMed Central

    Brooun, Alexei; Foster, Scott A.; Chrencik, Jill E.; Chien, Ellen Y.T.; Kolatkar, Anand R.; Streiff, Markus; Ramage, Paul; Widmer, Hans; Weckbecker, Gisbert; Kuhn, Peter

    2007-01-01

    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. PMID:17275330

  6. 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.

  7. Production and Partial Purification of Alpha Amylase from Bacillus subtilis (MTCC 121) Using Solid State Fermentation

    PubMed Central

    Raul, Dibyangana; Mukhopadhyay, Suchita; Kumar Das, Shrayan; Gupta, Suvroma

    2014-01-01

    Amylase is an enzyme that catalyzes the breakdown of starch into sugars and plays a pivotal role in a variety of areas like use as digestives, for the production of ethanol and high fructose corn syrup, detergents, desiring of textiles, modified starches, hydrolysis of oil-field drilling fluids, and paper recycling. In the present work, solid state fermentation (SSF) for α-amylase production has been used in lieu of submerged fermentation (SmF) due to its simple technique, low capital investment, lower levels of catabolite repression, and better product recovery. Bacillus subtilis has been well known as producer of alpha amylase and was tested using solid state fermentation for 48 hours at 37°C with wheat bran as substrate. Comparison between different fermentation hours demonstrated high yield of alpha amylase after 48 hours. This alpha amylase has optimum pH and temperature at 7.1 and 40°C, respectively. With the goal to purify alpha amylase, 30–70% (NH4)2SO4 cut concentrated the amylase activity threefold with respect to crude fermented extract. This was verified in quantitative DNS assay method as well as in zymogram gel profile. The exact molecular weight of the amylase is yet to be determined with the aid of other protein purification techniques. PMID:24672727

  8. Production and Partial Purification of Alpha Amylase from Bacillus subtilis (MTCC 121) Using Solid State Fermentation.

    PubMed

    Raul, Dibyangana; Biswas, Tania; Mukhopadhyay, Suchita; Kumar Das, Shrayan; Gupta, Suvroma

    2014-01-01

    Amylase is an enzyme that catalyzes the breakdown of starch into sugars and plays a pivotal role in a variety of areas like use as digestives, for the production of ethanol and high fructose corn syrup, detergents, desiring of textiles, modified starches, hydrolysis of oil-field drilling fluids, and paper recycling. In the present work, solid state fermentation (SSF) for α -amylase production has been used in lieu of submerged fermentation (SmF) due to its simple technique, low capital investment, lower levels of catabolite repression, and better product recovery. Bacillus subtilis has been well known as producer of alpha amylase and was tested using solid state fermentation for 48 hours at 37°C with wheat bran as substrate. Comparison between different fermentation hours demonstrated high yield of alpha amylase after 48 hours. This alpha amylase has optimum pH and temperature at 7.1 and 40°C, respectively. With the goal to purify alpha amylase, 30-70% (NH4)2SO4 cut concentrated the amylase activity threefold with respect to crude fermented extract. This was verified in quantitative DNS assay method as well as in zymogram gel profile. The exact molecular weight of the amylase is yet to be determined with the aid of other protein purification techniques.

  9. Purification, characterization and production optimization of a vibriocin produced by mangrove associated Vibrio parahaemolyticus

    PubMed Central

    Balakrishnan, Baskar; Ranishree, Jayappriyan Kothilmozhian; Thadikamala, Sathish; Panchatcharam, Prabakaran

    2014-01-01

    Objective To identify a potential bacterium which produces antimicrobial peptide (vibriocin), and its purification, characterization and production optimization. The bacteria subjected in the study were isolated from a highly competitive ecological niche of mangrove ecosystem. Methods The bacterium was characterized by phenotype besides 16S rRNA gene sequence analysis. The antibacterial activity was recognised by using agar well diffusion method. The vibriocin was purified using ammonium sulphate precipitation, butanol extraction, gel filtration chromatography, ion-exchange chromatography and subsequently, by HPLC. Molecular weight of the substance identified in SDS-PAGE. Production optimization performed according to Taguchi's mathematical model using 6 different nutritional parameters as variables. Results The objective bacterium was identified as Vibrio parahaemolyticus. The vibriocin showed 18 KDa of molecular mass with mono peptide in nature and highest activity against pathogenic Vibrio harveyi. The peptide act stable in a wide range of pH, temperature, UV radiation, solvents and chemicals utilized. An overall ∼20% of vibriocin production was improved, and was noticed that NaCl and agitation speed played a vital role in secretion of vibriocin. Conclusion The vibriocin identified here would be an effective alternative for chemically synthesized drugs for the management of Vibrio infections in mariculture industry. PMID:25182547

  10. 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.

  11. Automated purification of Borrelia burgdorferi s.l. PCR products with KingFisher™ magnetic particle processor prior to genome sequencing

    NASA Astrophysics Data System (ADS)

    Mäkinen, Johanna; Marttila, Harri; Viljanen, Matti K.

    2001-01-01

    Borrelia burgdorferi sensu lato genospecies were differentiated by PCR-based sequencing of the borrelial flagellin gene. To evaluate the usefulness of KingFisher™ magnetic particle processor in PCR product purification, borrelia PCR products were purified with KingFisher™ magnetic particle processor prior to cycle sequencing and the quality of the sequence data received was analyzed. KingFisher was found to offer a rapid and reliable alternative for borrelial PCR product purification.

  12. Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems.

    PubMed

    Parales, R E; Huang, R; Yu, C-L; Parales, J V; Lee, F K N; Lessner, D J; Ivkovic-Jensen, M M; Liu, W; Friemann, R; Ramaswamy, S; Gibson, D T

    2005-07-01

    The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.

  13. Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

    PubMed Central

    Parales, R. E.; Huang, R.; Yu, C.-L.; Parales, J. V.; Lee, F. K. N.; Lessner, D. J.; Ivkovic-Jensen, M. M.; Liu, W.; Friemann, R.; Ramaswamy, S.; Gibson, D. T.

    2005-01-01

    The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an α3β3 hexamer. The apparent Km of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM−1 min−1 for 2NT and 1.2 μM−1 min−1 for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained. PMID:16000792

  14. Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter

    PubMed Central

    Rouse, Sarah L.; Hawthorne, Wlliam J.; Lambert, Sebastian; Morgan, Marc L.; Hare, Stephen A.; Matthews, Stephen

    2016-01-01

    Bacteria often produce extracellular amyloid fibres via a multi-component secretion system. Aggregation-prone, unstructured subunits cross the periplasm and are secreted through the outer membrane, after which they self-assemble. Here, significant progress is presented towards solving the high-resolution crystal structure of the novel amyloid transporter FapF from Pseudomonas, which facilitates the secretion of the amyloid-forming polypeptide FapC across the bacterial outer membrane. This represents the first step towards obtaining structural insight into the products of the Pseudomonas fap operon. Initial attempts at crystallizing full-length and N-terminally truncated constructs by refolding techniques were not successful; however, after preparing FapF106–430 from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 Å resolution. These crystals belonged to the monoclinic space group C121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 Å, α = γ = 90, β = 96.32° and three monomers in the asymmetric unit. It was found that the switch to complete detergent exchange into C8E4 was crucial for forming well diffracting crystals, and it is suggested that this combined with limited proteolysis is a potentially useful protocol for membrane β-barrel protein crystallography. The three-dimensional structure of FapF will provide invaluable information on the mechanistic differences of biogenesis between the curli and Fap functional amyloid systems. PMID:27917837

  15. Current strategies for protein production and purification enabling membrane protein structural biology.

    PubMed

    Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

    2016-12-01

    Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

  16. Water purification systems: a comparative analysis based on the occurrence of disinfection by-products.

    PubMed

    Gibbons, J; Laha, S

    1999-09-01

    Trihalomethanes (THMs) are halogenated hydrocarbons, and are by-products of the chlorination of drinking water. Most THMs are formed in drinking water when chlorine reacts with naturally occurring organic substances such as decomposing plant and animal materials. Risks for certain types of cancer are now being correlated with the presence of disinfection by-products (DBPs). The present research uses gas chromatography to analyze the presence and levels of THMs in drinking water samples from a variety of sources. These include (1) municipal drinking water from two south Florida counties; (2) two brands of bottled water; (3) untreated residential well water; and (4) municipal tap water passed through additional water purification systems. The results are summarized in a tabular format, and the compliance of each water with existing US EPA-mandated standards is examined. General conclusions from this study are that all the waters tested complied with federal regulations regarding THM levels, properly functioning home filtration units may be quite effective in further reducing DBP concentrations and, as expected, non-chlorinated waters such as bottled water and residential well water contain lower THM levels.

  17. Purification of human ceruloplasmin as a by-product of C1-inhibitor.

    PubMed

    Kouoh Elombo, F; Radosevich, M; Poulle, M; Descamps, J; Chtourou, S; Burnouf, T; Catteau, J P; Bernier, J L; Cotelle, N

    2000-12-01

    Human ceruloplasmin (Cp) has been purified from cryoprecipitate-poor plasma as a by-product of the C1-inhibitor production chain. Highly purified Cp was obtained by subsequent ion-exchange chromatography on sulfate-Fractogel EMD and TMAE-Fractogel EMD. Treatments for viral safety included application of the solvent-detergent method and two nanofiltration steps using 35- and 15-nm pore size filters at the end of the process. Overall antigen yield was 95 (+/-5) %. Purified human ceruloplasmin was studied by electron spin resonance (ESR) to characterize its different types of copper complexes and to check its antioxidant properties. We distinguished three types of complexes: one type-2 Cu(II) with g// = 2.25 and A// = 180 G and two type-I Cu(II) exhibiting different narrow hyperfine splitting (A// = 72 G and A// = 90 G) with close g// (2.20 and 2.21). Purified Cp has a specific activity of 24.5+/-0.2 mU/mg of proteins. This process provides a method for Cp purification that could be easily integrated into modern plasma fractionation.

  18. Purification, crystallization and preliminary X-ray crystallographic analysis of a rice Rac/Rop GTPase, OsRac1

    PubMed Central

    Kosami, Ken-ichi; Ohki, Izuru; Hayashi, Kokoro; Tabata, Ryo; Usugi, Sayaka; Kawasaki, Tsutomu; Fujiwara, Toshimichi; Nakagawa, Atsushi; Shimamoto, Ko; Kojima, Chojiro

    2014-01-01

    Small GTPases regulate a large variety of key cellular processes. Plant small Rac/Rop GTPases have recently received broad attention as it is becoming clear that these enzymes regulate various plant cellular processes. OsRac1, a rice Rac/Rop protein, is a key regulator of reactive oxygen species (ROS) production and induces immune responses. Although four structures of plant small GTPases have been reported, all of these were of the inactive form. Here, OsRac1 was purified and co-crystallized with the GTP analogue 5′-guanylyl imidodiphos­phate (GMPPNP). The crystal belonged to space group P212121 and a complete data set was collected to 1.9 Å resolution. PMID:24419631

  19. Crystal Engineering: From Molecules to Products

    ERIC Educational Resources Information Center

    Doherty, Michael F.

    2006-01-01

    Particle production and solids processing are essential components of the contemporary process industries. Crystalline solids represent a large and important segment of this manufacturing sector. Chemical engineers, especially in the United States, have historically abandoned this subject, leaving it to pharmacists, physical chemists, material…

  20. 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

  1. 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.

  2. Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

    PubMed

    Armendáriz-Borunda, Juan; Bastidas-Ramírez, Blanca Estela; Sandoval-Rodríguez, Ana; González-Cuevas, Jaime; Gómez-Meda, Belinda; García-Bañuelos, Jesús

    2011-11-01

    Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture.

  3. Production and purification of recombinant enteropeptidase expressed in an insect-baculovirus cell system.

    PubMed

    Azhar, Mahammad; Somashekhar, R

    2015-01-01

    Enteropeptidase (EC 3.4.21.9) is the glycoprotein enzyme in the small intestine that triggers the activation of the zymogens in pancreatic juice by converting trypsinogen into trypsin. Because of its physiological significance, there have been many studies on the expression, purification, and characterization of enteropeptidase from different species. The baculovirus expression system has been commonly used in research communities and scientific industries for the production of high levels of recombinant proteins, which require posttranslational modifications for functional activity. In the present study, we isolated bovine enteropeptidase catalytic subunit gene from Bos taurus indicus (GenBank accession no. KC756844), and cloned it in pFast Bac HT "A" baculovirus expression donor vector, under the polyhedrin promoter. Recombinant bovine enteropeptidase was expressed in SF-9 insect cells with high expression levels. Recombinant enteropeptidase was purified using Ni-NTA affinity chromatography. A 6-mg quantity of pure active protein was obtained from 100 mL culture using this approach. Its activity and kinetic parameters were determined by cleavage of its fluorogenic substrate Gly-(Asp) 4-Lys-β-naphthylamide. The recombinant bovine enteropeptidase showed a K m value of 0.75 ± 0.02 mM with K cat 25 ± 1 s.

  4. Production, purification and application of polysaccharide-based bioflocculant by Paenibacillus mucilaginosus.

    PubMed

    Tang, Jiayi; Qi, Suijian; Li, Zhigang; An, Qun; Xie, Mingquan; Yang, Bo; Wang, Yonghua

    2014-11-26

    The production and purification of polysaccharide-based bioflocculants (PSBs) by Paenibacillus mucilaginosus GIM1.16 in metal ion-supplemented medium and basal medium were evaluated. Three purified PSB1-1, PSB2-1 and PSB3-1 possessed different monosaccharide composition and their molecular weights were 2.53 × 10(6), 7.77 × 10(6) and 13.2 × 10(6)Da, respectively. FT-IR spectrometry indicated the presence of hydroxyl, carboxyl and phosphate groups in the three samples. Scanning electron microscopy showed that they had linear structure. The potential of these PSBs on wastewater treatment was evaluated. Among them, PSB1-1 exhibited the best performance, as it had high flocculating activities (above 94%) at 0.5-4 mg/L and could achieve high flocculating activities (above 97%) in the kaolin suspensions of pH 3-9. PSB1-1 was the key factor that might explain the enhanced flocculating activity of the supernatant from metal ion-supplemented medium. The performance of PSB1-1 on industrial wastewater was also satisfactory. PSB1-1 might be a good candidate as bioflocculant.

  5. Production, Purification, and in Vitro Evaluation of the Prebiotic Potential of Arabinoxylooligosaccharides from Brewer's Spent Grain.

    PubMed

    Gómez, Belén; Míguez, Beatriz; Veiga, Adán; Parajó, Juan Carlos; Alonso, José Luís

    2015-09-30

    Brewer's spent grain (BSG) samples were subjected to a two-step aqueous processing (starch extraction and autohydrolysis) in order to assess their potential as a raw material for obtaining a mixture of arabinoxylooligosaccharides (AXOS) suitable to be use as prebiotics for elderly. After hydrothermal treatment, the liquors were refined by a sequence of purification and conditioning steps including membrane filtration, enzymatic hydrolysis, and ion exchange. The presence of both substituted (degree of polimerization (DP) = 2-10) and unsubstituted (DP = 2-16) oligosaccharides made up of xylose and arabinose (AXOS) were confirmed in purified mixtures (in which total OS content = 84% w/w) by using chromatographic techniques and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Finally, AXOS were evaluated for their prebiotic activity by in vitro fermentation assays using fecal inocula from elderly people, demonstrating that AXOS were slightly better substrates than FOS, in terms of bacterial population shifts as in the production of SCFA.

  6. Analysis and Purification of Bioactive Natural Products: The AnaPurNa Study

    PubMed Central

    2012-01-01

    Based on a meta-analysis of data mined from almost 2000 publications on bioactive natural products (NPs) from >80 000 pages of 13 different journals published in 1998–1999, 2004–2005, and 2009–2010, the aim of this systematic review is to provide both a survey of the status quo and a perspective for analytical methodology used for isolation and purity assessment of bioactive NPs. The study provides numerical measures of the common means of sourcing NPs, the chromatographic methodology employed for NP purification, and the role of spectroscopy and purity assessment in NP characterization. A link is proposed between the observed use of various analytical methodologies, the challenges posed by the complexity of metabolomes, and the inescapable residual complexity of purified NPs and their biological assessment. The data provide inspiration for the development of innovative methods for NP analysis as a means of advancing the role of naturally occurring compounds as a viable source of biologically active agents with relevance for human health and global benefit. PMID:22620854

  7. Production, purification and biological characterization of mono-PEGylated anti-IL-17A antibody fragments.

    PubMed

    Koussoroplis, Salome-Juliette; Heywood, Sam; Uyttenhove, Catherine; Barilly, Céline; Van Snick, Jacques; Vanbever, Rita

    2013-09-15

    The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥ 20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab')2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies.

  8. 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

  9. 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.

  10. Preparative Purification of Liriodendrin from Sargentodoxa cuneata by Macroporous Resin.

    PubMed

    Li, Di-Hua; Wang, Yan; Lv, Yuan-Shan; Liu, Jun-Hong; Yang, Lei; Zhang, Shu-Kun; Zhuo, Yu-Zhen

    2015-01-01

    The preparative purification of liriodendrin from Sargentodoxa cuneata using macroporous resin combined with crystallization process was evaluated. The properties of adsorption/desorption of liriodendrin on eight macroporous resins were investigated systematically. X-5 resin was selected as the most suitable medium for liriodendrin purification. The adsorption of liriodendrin on X-5 resin fitted well with the pseudo-second-order kinetic model and Langmuir isotherm model. Dynamic adsorption/desorption tests were performed using a glass column packed with X-5 resin to optimize the separation process of liriodendrin. After one treatment with X-5 resin, the content of liriodendrin in the product was increased 48.73-fold, from 0.85% to 41.42%, with a recovery yield of 88.9%. 97.48% liriodendrin was obtained by further crystallization and determined by HPLC. The purified product possessed strong antioxidant activity. In conclusion, purification of liriodendrin might expend its further pharmacological researches and further applications in pharmacy.

  11. DOE Hydrogen, Fuel Cells and Infrastructure Technologies Program Integrated Hydrogen Production, Purification and Compression System

    SciTech Connect

    Tamhankar, Satish; Gulamhusein, Ali; Boyd, Tony; DaCosta, David; Golben, Mark

    2011-06-30

    The project was started in April 2005 with the objective to meet the DOE target of delivered hydrogen of <$1.50/gge, which was later revised by DOE to $2-$3/gge range for hydrogen to be competitive with gasoline as a fuel for vehicles. For small, on-site hydrogen plants being evaluated at the time for refueling stations (the 'forecourt'), it was determined that capital cost is the main contributor to the high cost of delivered hydrogen. The concept of this project was to reduce the cost by combining unit operations for the entire generation, purification, and compression system (refer to Figure 1). To accomplish this, the Fluid Bed Membrane Reactor (FBMR) developed by MRT was used. The FBMR has hydrogen selective, palladium-alloy membrane modules immersed in the reformer vessel, thereby directly producing high purity hydrogen in a single step. The continuous removal of pure hydrogen from the reformer pushes the equilibrium 'forward', thereby maximizing the productivity with an associated reduction in the cost of product hydrogen. Additional gains were envisaged by the integration of the novel Metal Hydride Hydrogen Compressor (MHC) developed by Ergenics, which compresses hydrogen from 0.5 bar (7 psia) to 350 bar (5,076 psia) or higher in a single unit using thermal energy. Excess energy from the reformer provides up to 25% of the power used for driving the hydride compressor so that system integration improved efficiency. Hydrogen from the membrane reformer is of very high, fuel cell vehicle (FCV) quality (purity over 99.99%), eliminating the need for a separate purification step. The hydride compressor maintains hydrogen purity because it does not have dynamic seals or lubricating oil. The project team set out to integrate the membrane reformer developed by MRT and the hydride compression system developed by Ergenics in a single package. This was expected to result in lower cost and higher efficiency compared to conventional hydrogen production technologies. The

  12. [Production of mutagenic compounds during the water purification treatment of surface water].

    PubMed

    Gilli, G; Carraro, E; Ferrara, A

    1991-01-01

    In the last years many studies have reported the presence of mutagenic/carcinogenic compounds in treated waters. These substances can be present in raw water, but are also produced during drinking water purification. Mutagens are formed as by-products of chemical reactions between oxidants/disinfectants used in treatments and organic load of the raw water (humic and fulvic acids). The aim of this study was to evaluate the production of mutagenic substances during the main phases of the Po river water treatment ("PO3" plant) in Turin. Water samples (50 litres), collected from February 1989 to August 1990, were concentrated with XAD-2/XAD-8 resins mixture. Extracts were tested for mutagenicity at different doses (1, 2.5, 5 and 10 litres) by Ames Salmonella assay, using TA 100 and TA 98 strains, without microsome fraction (S9). Raw water was rarely mutagenic while, in particular at the highest doses (5 and 10 litres), sometimes showed toxic effect. After ozonation treatment only few samples were mutagenic with TA 100 strain, while 43% of the samples were mutagenic with TA 98. The following treatment of clariflocculation and chlorination with NaClO produced mutagens in 95% of the samples assayed with TA 100 and in 85% of the samples assayed with TA 98. The next GAC/sand filtration step seems to reduce the mutagenic load produced in the previous phases. Finally, drinking water after chlorination with ClO2 showed weak mutagenicity at 1 litre dose (26% and 21% of positive samples with TA 100 and TA 98 respectively) and this effect increased at the higher dosages.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

    PubMed

    Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

    2013-11-01

    Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pHproduct variants using the combined pH and salt based elution gradient and removal of the host cell impurities in flow-through are the key novel features of the developed multimodal chromatographic purification step.

  14. 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.

  15. Cloning, expression, purification and crystallization of a transcriptional regulatory protein (Rv3291c) from Mycobacterium tuberculosis H37Rv.

    PubMed

    Shrivastava, Tripti; Kumar, Sandeep; Ramachandran, Ravishankar

    2004-10-01

    Rv3291c, the translational product of the Mycobacterium tuberculosis Rv3291c gene, is an 18 kDa protein. It is a putative transcriptional regulatory protein belonging to the leucine-responsive regulatory protein/asparagine synthase C (Lrp/AsnC) family, which are proteins that have been identified in archaea and bacteria. Rv3291c probably plays a significant role during the persistent/latent phase of M. tuberculosis, as supported by its up-regulation several-fold during this stage. Orthorhombic crystals of recombinant Rv3291c have been grown from trisodium citrate dihydrate-buffered solutions containing monoammonium dihydrogen phosphate. Diffraction data extending to 2.7 A have been collected from a single crystal with unit-cell parameters a = 99.6, b = 100.7, c = 100.6 A. Assuming an octamer in the asymmetric unit results in a Matthews coefficient (VM) of 1.75 A3 Da(-1), corresponding to a solvent content of about 30%.

  16. Production and purification of refolded recombinant Plasmodium falciparum beta-ketoacyl-ACP reductase from inclusion bodies.

    PubMed

    Karmodiya, Krishanpal; Srivastav, Ratnesh Kumar; Surolia, Namita

    2005-07-01

    A recombinant form of Plasmodium falciparum beta-ketoacyl-ACP reductase (PfFabG) was overexpressed in Escherichia coli BL-21 codon plus (DE3). The resulting insoluble inclusion bodies were separated from cellular debris by extensive washing with buffer containing 0.05% Tween 20 and solubilized by homogenization with 8 M urea. Attempts to refold PfFabG from solubilized inclusion bodies employing Rotofor (separation based on different pIs of proteins in a mixture) followed by Ni(2+) or cation exchange chromatography were not successful either by bringing down the urea concentration instantaneously, stepwise, or by dialysis. Denatured PfFabG was therefore initially purified by cation exchange chromatography and was then correctly refolded at a final concentration of 100-200 microg/ml in a 20 mM Na-acetate buffer, pH 5.3, with 300 mM NaCl, 10% glycerol, and 0.05% Tween 20. The protein was found to be properly folded only in the presence of the cofactor NADPH and salt at a concentration 300 mM by drop dilution method at 2-8 degrees C for 12 h. The purified final product was >98% pure by denaturing gel electrophoresis. The purified protein was biologically active in a standard enzymatic assay using acetoacetyl-CoA as a substrate. The enzyme was found to be stable up to fourth day of purification and glycerol was found to stabilize enzyme activity for several weeks, during storage. This effort paves the way for elucidation of the structure-function correlations for PfFabG as well as exploration of the enzyme for developing inhibitors against it for combating malaria.

  17. Purification, crystallization and preliminary X-ray diffraction analysis of aspartate semialdehyde dehydrogenase (Rv3708c) from Mycobacterium tuberculosis

    SciTech Connect

    Vyas, Rajan; Panjikar, Santosh; Kishan, K. V. Radha; Tewari, Rupinder; Weiss, Manfred S.

    2008-03-01

    The enzyme aspartate semialdehyde dehydrogenase from M. tuberculosis has been expressed, purified and crystallized in two different crystal forms. Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B.

  18. Production of Computationally Designed Small Soluble- and Membrane-Proteins: Cloning, Expression, and Purification.

    PubMed

    Tripathy, Barsa; Acharya, Rudresh

    2017-01-01

    This book chapter focuses on expression and purification of computationally designed small soluble proteins and membrane proteins that are ordinarily difficult to express in good amounts for experiments. Over-expression of such proteins can be achieved by using the solubility tag such as maltose binding protein (MBP), Thioredoxin (Trx), and Gultathione-S-transferase (GST) fused to the protein of interest. Here, we describe and provide the protocols for cloning, expression and purification of such proteins using the solubility tag.

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

    PubMed

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

    2001-02-01

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

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

    PubMed Central

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

    2001-01-01

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

  1. A simple and efficient purification platform for monoclonal antibody production based on chromatin-directed cell culture clarification integrated with precipitation and void-exclusion anion exchange chromatography.

    PubMed

    Chen, Quan; Abdul Latiff, Sarah Maria; Toh, Phyllicia; Peng, Xinying; Hoi, Aina; Xian, Mo; Zhang, Haibo; Nian, Rui; Zhang, Wei; Gagnon, Pete

    2016-10-20

    Protein A affinity chromatography, featured by its robustness and high-specificity, is still dominant as a first capture step for the purification of immunoglobulin G monoclonal antibodies (IgG mAbs). However, the material and operational costs of protein A are universally recognized as high, and its productivity is also limited as column mode. In order to overcome these limitations, industry is increasingly considering the use of non-protein A-based processes for IgG purification. In this study, sodium citrate precipitation (SCP) was developed as the primary purification step, and chromatin-directed cell culture clarification was demonstrated to significantly elevate the purification capability. Additional 0.05% (w/v) of Tween 20 was shown to effectively reduce the residual free antibody light chain (LC) during precipitation. The resuspended IgG was further polished by void-exclusion anion exchange chromatography (VEAX), which supported protein loading without buffer adjustment. The non-histone host cell protein (nh-HCP) content in the final product was about 5ppm and histone HCP below limit of detection (LOD). DNA was reduced to less than 1ppb, and aggregates/free LC less than 0.1%. The overall IgG recovery was 87.2%. A simple and efficient purification platform with only one-column step was therefore established, providing a more promising alternative to the current prevailing protein A-based purification platforms.

  2. Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

    SciTech Connect

    López-Jaramillo, F. J.; Pérez-Banderas, F.; Hernández-Mateo, F.; Santoyo-González, F.

    2005-04-01

    The feasibility of glycosylation post-purification has been demonstrated by introducing glucose into the model protein lysozyme via a novel reaction that is compatible with biological samples. The crystallization of glycoproteins is one of the challenges to be confronted by the crystallographic community in the frame of what is known as glycobiology. The state of the art for the crystallization of glycoproteins is not promising and removal of the carbohydrate chains is generally suggested since they are flexible and a source of heterogeneity. In this paper, the feasibility of introducing glucose into the model protein hen egg-white lysozyme via a post-purification glycosylation reaction that may turn any protein into a model glycoprotein whose carbohydrate fraction can be manipulated is demonstrated.

  3. 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.

  4. 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.

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

    PubMed Central

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

    2009-01-01

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

  6. 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.

  7. 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.

  8. Bioactivities, isolation and purification methods of polysaccharides from natural products: A review.

    PubMed

    Shi, Lei

    2016-11-01

    Polysaccharides play multiple roles and have extensive bioactivities in life process and an immense potential in healthcare, food and cosmetic industries, due to their therapeutic effects and relatively low toxicity. This review describes their major functions involved in antitumor, anti-virus, and anti-inflammatory bioactivities. Due to their enormous structural heterogeneity, the approaches for isolation and purification of polysaccharides are distinct from that of the other macromolecules such as proteins, etc. Yet, to achieve the homogeneity is the initial step for studies of polysaccharide structure, pharmacology, and its structure-activity relationships. According to the experiences accumulated by our lab and the published literatures, this review also introduces the methods widely used in isolation and purification of polysaccharides.

  9. Production and purification of xylooligosaccharides from oil palm empty fruit bunch fibre by a non-isothermal process.

    PubMed

    Ho, Ai Ling; Carvalheiro, Florbela; Duarte, Luís C; Roseiro, Luísa B; Charalampopoulos, Dimitris; Rastall, Robert A

    2014-01-01

    Oil palm empty fruit bunches (OPEFB) fibre, a by-product generated from non-woody, tropical perennial oil palm crop was evaluated for xylooligosaccharides (XOS) production. Samples of OPEFB fibre were subjected to non-isothermal autohydrolysis treatment using a temperature range from 150 to 220 °C. The highest XOS concentration, 17.6g/L which relayed from solubilisation of 63 g/100 g xylan was achieved at 210 °C and there was a minimum amount of xylose and furfural being produced. The chromatographic purification which was undertaken to purify the oligosaccharide-rich liquor resulted in a product with 74-78% purity, of which 83-85% was XOS with degree of polymerisation (DP) between 5 and 40.

  10. Purification of uranium alloys by differential solubility of oxides and production of purified fuel precursors

    DOEpatents

    McLean, W. II; Miller, P.E.

    1997-12-16

    A method is described for purifying metallic alloys of uranium for use as nuclear reactor fuels in which the metal alloy is first converted to an oxide and then dissolved in nitric acid. Initial removal of metal oxide impurities not soluble in nitric acid is accomplished by filtration or other physical means. Further purification can be accomplished by carbonate leaching of uranyl ions from the partially purified solution or using traditional methods such as solvent extraction. 3 figs.

  11. Purification of uranium alloys by differential solubility of oxides and production of purified fuel precursors

    DOEpatents

    McLean, II, William; Miller, Philip E.

    1997-01-01

    A method for purifying metallic alloys of uranium for use as nuclear reactor fuels in which the metal alloy is first converted to an oxide and then dissolved in nitric acid. Initial removal of metal oxide impurities not soluble in nitric acid is accomplished by filtration or other physical means. Further purification can be accomplished by carbonate leaching of uranyl ions from the partially purified solution or using traditional methods such as solvent extraction.

  12. A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides

    PubMed Central

    Hoarau, Marie; Hureau, Christelle; Faller, Peter; Gras, Emmanuel; André, Isabelle; Remaud-Siméon, Magali

    2016-01-01

    An improved production and purification method for Alzheimer’s disease related methionine-modified amyloid-β 1–40 and 1–42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-β peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/ neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies. PMID:27532547

  13. High-level expression, purification and production of the fungal immunomodulatory protein-gts in baculovirus-infected insect larva.

    PubMed

    Wu, Tzong-Yuan; Chen, Hsin-An; Li, Feng-Yin; Lin, Ching-Ting; Wu, Chi-Ming; Hsieh, Feng-Chia; Tzen, Jason Tze-Cheng; Hsieh, Sheng-Kuo; Ko, Jiunn-Liang; Jinn, Tzyy-Rong

    2013-02-01

    Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP(bbx) secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 10⁹ cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.

  14. 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.

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

    SciTech Connect

    Ohtsuki, Takayuki; Ohshima, Shigeru; Uchida, Akira

    2007-09-01

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

  16. Improved strategy for recombinant production and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity.

    PubMed

    Panteleev, Pavel V; Ovchinnikova, Tatiana V

    2017-01-01

    Here, we report an efficient procedure for recombinant production and purification of tachyplesin I (THI) with a final yield of 17 mg/L of the culture medium. The peptide was expressed in Escherichia coli as a part of the thioredoxin fusion protein. With the use of soluble expression followed by immobilized metal-ion affinity chromatography, the recombinant protein cleavage and reversed-phase high-performance liquid chromatography, a yield of THI did not exceed 6.5 mg/L of the culture medium. Further optimization studies were carried out to improve the protein expression level and simplify purification procedure of the target peptide. To achieve better yield of the peptide, we used high-cell-density bacterial expression. The formed inclusion bodies were highly enriched with the fusion protein, which allowed us to perform direct chemical cleavage of the inclusion bodies solubilized in 6 M guanidine-HCl with subsequent selective precipitation of proteins with trifluoroacetic acid. This enabled us to avoid an extra step of purification by immobilized metal-ion affinity chromatography. The developed procedure has made it possible to obtain biologically active THI and was used for screening a number of its mutant analogs. As a result, several selective and nonhemolytic analogs were developed. Significant reduction in hemolytic activity without losing antimicrobial activity was achieved by substitution of tyrosine or isoleucine residue in the β-turn region of the molecule with hydrophilic serine. The present study affords further insight into molecular mechanism of antimicrobial action of tachyplesin and gains a better understanding of structure-activity relationships in its analogs. This is aimed at searching for novel antibiotics on the basis of antimicrobial peptides with reduced cytotoxicity.

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

    SciTech Connect

    Tripathi, Sarvind Mani; Ramachandran, Ravishankar

    2008-05-01

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

  18. A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis.

    PubMed

    Okvist, Mats; Sasso, Severin; Roderer, Kathrin; Kast, Peter; Krengel, Ute

    2009-10-01

    Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM-MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM-MtDS complex diffracted to 1.6 and 2.1 A resolution, respectively.

  19. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the TIR domain from the Brucella melitensis TIR-domain-containing protein TcpB.

    PubMed

    Alaidarous, Mohammed; Ve, Thomas; Ullah, M Obayed; Valkov, Eugene; Mansell, Ashley; Schembri, Mark A; Sweet, Matthew J; Kobe, Bostjan

    2013-10-01

    In mammals, Toll-like receptors (TLRs) recognize conserved microbial molecular signatures and induce an early innate immune response in the host. TLR signalling is mediated by interactions between the cytosolic TIR (Toll/interleukin-1 receptor) domains of the receptor and the adaptor proteins. Increasingly, it is apparent that pathogens target this interaction via pathogen-expressed TIR-domain-containing proteins to modulate immune responses. A TIR-domain-containing protein TcpB has been reported in the pathogenic bacterium Brucella melitensis. Studies have shown that TcpB interferes with the TLR2 and TLR4 signalling pathways to inhibit TLR-mediated inflammatory responses. Such interference may involve TIR-TIR-domain interactions between bacterial and mammalian proteins, but there is a lack of information about these interactions at the molecular level. In this study, the cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the protein construct corresponding to the TIR domain of TcpB (residues 120-250) are reported. The crystals diffracted to 2.6 Å resolution, have the symmetry of the monoclinic space group P2₁ and are most likely to contain four molecules in the asymmetric unit. The structure should help in understanding the molecular basis of how TcpB affects the innate immunity of the host.

  20. Potential productivity benefits of float-zone versus Czochralski crystal growth

    NASA Technical Reports Server (NTRS)

    Abe, T.

    1985-01-01

    Efficient mass production of single-crystal silicon is necessary for the efficient silicon solar arrays needed in the coming decade. However, it is anticipated that there will be difficulty growing such volumes of crystals using conventional Czochralski (Cz) methods. While the productivity of single crystals might increase with a crystal diameter increase, there are two obstacles to the mass production of large diameter Czochralski crystals, the long production cycle due to slow growth rate and the high heat requirements of the furnaces. Also counterproductive would be the large resistivity gradient along the growth direction of the crystals due to impurity concentration. Comparison between Float zone (FZ) and Cz crystal growth on the basis of a crystal 150 mm in diameter is on an order of two to four times in favor of the FZ method. This advantage results from high growth rates and steady-state growth while maintaining a dislocation-free condition and impurity segregation.

  1. Purification, crystallization and preliminary X-ray analysis of FliT, a bacterial flagellar substrate-specific export chaperone

    PubMed Central

    Kinoshita, Miki; Yamane, Midori; Matsunami, Hideyuki; Minamino, Tohru; Namba, Keiichi; Imada, Katsumi

    2009-01-01

    The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD4C2 complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3121 or P3221 and diffracted to 3.2 Å resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination. PMID:19652350

  2. Cloning, expression, purification, crystallization and preliminary X-ray studies of epoxide hydrolases A and B from Mycobacterium tuberculosis

    SciTech Connect

    Biswal, Bichitra K.; Garen, Grace; Cherney, Maia M.; Garen, Craig; James, Michael N. G.

    2006-02-01

    Epoxide hydrolases A (Rv3617) and B (Rv1938), detoxification enzymes from M. tuberculosis, have been cloned, expressed, purified and crystallized. Crystals of Rv3617 and Rv1938 diffracted to 3.0 and 2.1 Å resolution, respectively. Mycobacterium tuberculosis epoxide hydrolases A and B, corresponding to open reading frames Rv3617 and Rv1938, are detoxification enzymes against epoxides. The recombinant forms of these enzymes have been expressed in Escherichia coli and purified to homogeneity. Diffraction-quality crystals of Rv3617 and Rv1938 were obtained by the hanging-drop vapour-diffusion technique. Crystals of Rv3617 and Rv1938 diffracted to 3.0 and 2.1 Å resolution, respectively, at the ALS synchrotron at Berkeley, CA, USA.

  3. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  5. 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.

  6. 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%.

  7. Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein.

    PubMed

    Sommer, Benjamin; Friehs, Karl; Flaschel, Erwin; Reck, Michael; Stahl, Frank; Scheper, Thomas

    2009-03-25

    Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to alpha-1,4-glucans.

  8. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38)

    SciTech Connect

    Vasudevan, Dileep; Gopalan, Gayathri; He, Zengyong; Luan, Sheng; Swaminathan, Kunchithapadam

    2005-12-01

    Crystallization of Arabidopsis thaliana cyclophilin 38. The crystal diffracts X-rays to 2.5 Å resolution. AtCyp38 is one of the highly divergent multidomain cyclophilins from Arabidopsis thaliana. A recombinant form of AtCyp38 (residues 83–437) was expressed in Escherichia coli and purified to homogeneity. The protein was crystallized using the vapour-batch technique with PEG 6000 and t-butanol as precipitants. Crystals of recombinant AtCyp38 diffracted X-rays to better than 2.5 Å resolution at 95 K using a synchrotron-radiation source. The crystal belongs to the C-centred orthorhombic space group C222{sub 1}, with unit-cell parameters a = 58.2, b = 95.9, c = 167.5 Å, and contains one molecule in the asymmetric unit. The selenomethionine derivative of the AtCyp38 protein was overexpressed, purified and crystallized in the same space group and data were collected to 3.5 Å at the NSLS synchrotron. The structure is being solved by the MAD method.

  9. General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays.

    PubMed

    Tomasiak, Thomas M; Pedersen, Bjørn P; Chaudhary, Sarika; Rodriguez, Andrew; Colmanares, Yaneth Robles; Roe-Zurz, Zygy; Thamminana, Sobha; Tessema, Meseret; Stroud, Robert M

    2014-08-01

    This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable.

  10. 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

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

    SciTech Connect

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

    2007-04-01

    A CcdB homologue from V. fischeri was overexpressed in E. coli and purified. The free protein was crystallized, as were its complexes with fragments of E. coli and V. fischeri gyrase and with the F-plasmid CcdA C-terminal domain. The ccd toxin–antitoxin module from the Escherichia coli F plasmid has a homologue on the Vibrio fischeri integron. The homologue of the toxin (CcdB{sub Vfi}) was crystallized in two different crystal forms. The first form belongs to space group I23 or I2{sub 1}3, 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 CcdB{sub Vfi} with the GyrA14{sub Vfi} fragment of V. fischeri gyrase crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, 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 GyrA14{sub Ec} 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 CcdB{sub Vfi} and part of the F-plasmid antitoxin CcdA{sub F} crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.9, b = 62.6, c = 82.0 Å, and diffracts to 1.9 Å resolution.

  12. 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

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

    PubMed

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

    2012-06-01

    Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP(5) 2-K) is a key enzyme that catalyzes the synthesis of phytic acid (IP(6)) from inositol 1,3,4,5,6-pentakisphosphate (IP(5)) and ATP. The first structure of IP(5) 2-K, that from Arabidopsis thaliana, has been solved previously; it only crystallized in the presence of inositol, either the substrate IP(5) or the product IP(6), and failed to crystallize in its free state (without inositol). Based on structural analysis, a point mutation of IP(5) 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 IP(5) 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 P2(1)2(1)2, 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.

  14. 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%.

  15. 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

  16. Design, expression, and purification of a Flaviviridae polymerase using a high-throughput approach to facilitate crystal structure determination

    PubMed Central

    Choi, Kyung H.; Groarke, James M.; Young, Dorothy C.; Rossmann, Michael G.; Pevear, Daniel C.; Kuhn, Richard J.; Smith, Janet L.

    2004-01-01

    Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA-dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695-residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical “core,” although overall sequence identity is low. Eighty-four expression clones of the BVDV polymerase were designed by fine-sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high-throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96-well format, and two of these proteins were successfully crystallized. PMID:15388860

  17. 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.

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

    SciTech Connect

    Boehmer, Thomas; Schwartz, Thomas U.

    2007-09-01

    A heterodimeric complex consisting of the C-terminal domains of human nucleoporins Nup107 and Nup133 was purified and crystallized and complete diffraction data sets were collected. The nuclear pore complex (NPC), the sole gateway of traffic between the nucleus and the cytoplasm, is built up from multiple copies of about 30 proteins collectively termed nucleoporins (nups). Nups are organized into distinct subcomplexes. Nup107 and Nup133 are members of the essential Nup107–160 subcomplex, a component of the central NPC architecture. A dimeric complex of the C-terminal domains of human Nup107 and Nup133 was expressed from a bicistronic vector in Escherichia coli, purified and crystallized in two different crystal forms. Crystals grown in the presence of 18–22% PEG 3350 belong to space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 2.9 Å. Native and seleno-l-methionine-derivative crystals grown in the presence of 1.1 M sodium malonate belong to space group C2 and diffracted to 2.55 and 2.9 Å, respectively. Structure determination of this complex will give the first insights into the protein–protein interactions within a core module of the NPC.

  19. 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.

  20. Neutron Production via a Pyroelectric Crystal without a Tip

    NASA Astrophysics Data System (ADS)

    Tornow, W.; Shafroth, S. M.; Brownridge, J. D.

    2007-04-01

    Recently, Naranjo et al.^1 and Geuther et al.^2 reported on the production of neutrons via the ^2H(d,n)^3He reaction using a pyroelectric crystal with a tungsten tip attached. Here we report that neutrons can also be produced with a simpler version. Our accelerator consisted of a 2.54 cm dia x 2.54 cm LiTaO3 crystal placed in D2 gas of 2 mTorr without a tip and without a deuterated foil. The D2 provided the projectiles and target atoms for the ^2H(d,n)^3He reaction. When the heated (by a Peltier heater/cooler) crystal was allowed to cool to room temperature, our 12.5 cm dia x 5 cm liquid scintillator based neutron detector equipped with neutron-gamma-ray pulse-shape discrimination electronics counted 6 neutrons per minute compared to a background rate of 2 events per minute. The neutron detector was shielded by about 6 mm of Pb from the very intense X-ray radiation (˜100 mR/h). The maximum ion energy and current were 200 keV and 3 nA, respectively. When H2 was substituted for D2, no neutron counts above background were detected. ^1B. Naranjo, J.K. Gimzewski, and S. Putterman, Nature 434, 115 (2005) ^2J. Geuther, Y. Danon, and F. Saglime, Phy. Rev. Lett. 96, 054803 (2006) *This work was supported in part by DOE grant DE-FG02-97ER41033.

  1. 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

  2. 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.

  3. Purification, crystallization and preliminary crystallographic studies of an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli.

    PubMed

    Abramson, J; Larsson, G; Byrne, B; Puustinen, A; Garcia-Horsman, A; Iwata, S

    2000-08-01

    Cytochrome bo(3) ubiquinol oxidase has been successfully purified for crystallization. Single crystals of this integral membrane protein diffract X-rays to 3.5 A resolution and belong to the orthorhombic space group C222(1). From the diffraction data, the unit-cell parameters were determined to be a = 91.3, b = 370.3, c = 232.4 A. The crystals have a solvent content of 59% and contain two molecules per asymmetric unit. A search model generated from the structures of cytochrome c oxidase from Paracoccus denitrificans and the extrinsic domain of cytochrome bo(3) ubiquinol oxidase from Escherichia coli was used for molecular-replacement studies, resulting in a solution with sensible molecular packing.

  4. 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.

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

    PubMed

    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

    Rice bifunctional alpha-amylase/subtilisin inhibitor (RASI) can inhibit both alpha-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 angstroms resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 angstroms. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%.

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

    PubMed

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

    2010-01-01

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

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

    SciTech Connect

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

    2007-07-01

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

  9. 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.

  10. 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.

  11. Purification, crystallization and preliminary X-ray diffraction studies of the arsenic repressor ArsR from Corynebacterium glutamicum

    PubMed Central

    Santha, Sangilimadan; Pandaranayaka, Eswari P. J.; Rosen, Barry P.; Thiyagarajan, Saravanamuthu

    2011-01-01

    ArsR is a member of the SmtB/ArsR family of metalloregulatory proteins that regulate prokaryotic arsenic-resistance operons. Here, the crystallization and preliminary X-ray diffraction studies of a cysteine-free derivative of ArsR from Corynebacterium glutamicum (CgArsR-C15/16/55S) are reported. CgArsR-C15/16/55S was expressed, purified, crystallized and X-ray diffraction data were collected to 1.86 Å resolution. The protein crystallized in a tetragonal space group (P4), with unit-cell parameters a = b = 41.84, c = 99.47 Å. PMID:22139180

  12. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyoxalase I from Leishmania infantum

    PubMed Central

    Barata, Lídia; Sousa Silva, Marta; Schuldt, Linda; da Costa, Gonçalo; Tomás, Ana M.; Ferreira, António E. N.; Weiss, Manfred S.; Ponces Freire, Ana; Cordeiro, Carlos

    2010-01-01

    Glyoxalase I (GLO1) is the first of the two glyoxalase-pathway enzymes. It catalyzes the formation of S-d-lactoyltrypanothione from the non-enzymatically formed hemithioacetal of methylglyoxal and reduced trypanothione. In order to understand its substrate binding and catalytic mechanism, GLO1 from Leishmania infantum was cloned, overexpressed in Escherichia coli, purified and crystallized. Two crystal forms were obtained: a cube-shaped form and a rod-shaped form. While the cube-shaped form did not diffract X-rays at all, the rod-­shaped form exhibited diffraction to about 2.0 Å resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 130.03, b = 148.51, c = 50.63 Å and three dimers of the enzyme per asymmetric unit. PMID:20445262

  13. Expression, purification, crystallization and preliminary X-ray analysis of the RecQ helicase catalytic core from Deinococcus radiodurans.

    PubMed

    Chen, Sheng-Chia; Huang, Chi-Hung; Yang, Chia-Shin; Chang, Chi-Huang; Kuan, Shu-Min; Chan, Nei-Li; Chen, Yeh

    2012-10-01

    The RecQ proteins are a highly conserved group of DNA helicases which play crucial roles in the maintenance of genome stability. DrRecQ from the radioresistant bacterium Deinococcus radiodurans is a special member of the RecQ family because it contains three Helicase-and-RNase-D-C-terminal (HRDC) domains at the C-terminus. The helicase catalytic core is essential for ATPase and DNA-unwinding activities. In this work, the helicase catalytic core of DrRecQ was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour diffusion method and X-ray diffraction data were collected to 2.9 Å resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.75, b = 95.61, c = 183.83 Å.

  14. 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)

  15. A gas circulation and purification system for gas-cell-based low-energy RI-beam production

    NASA Astrophysics Data System (ADS)

    Sonoda, T.; Tsubota, T.; Wada, M.; Katayama, I.; Kojima, T. M.; Reponen, M.

    2016-06-01

    A gas circulation and purification system was developed at the RIKEN Radioactive Isotope Beam Factory that can be used for gas-cell-based low-energy RI-beam production. A high-flow-rate gas cell filled with one atmosphere of buffer gas (argon or helium) is used for the deceleration and thermalization of high-energy RI-beams. The exhausted buffer gas is efficiently collected using a compact dry pump and returned to the gas cell with a recovery efficiency of >97%. The buffer gas is efficiently purified using two gas purifiers as well as collision cleaning, which eliminates impurities in the gas. An impurity level of one part per billion is achieved with this method.

  16. Purification and characterization of solvent tolerant lipase from Bacillus sp. for methyl ester production from algal oil.

    PubMed

    Sivaramakrishnan, Ramachandran; Incharoensakdi, Aran

    2016-05-01

    Lipase from Bacillus sp. isolated from the oil contaminated soil was purified by ammonium sulphate precipitation and ion-exchange chromatography with a 5.1-fold purification and 10.5% yield. SDS-PAGE analysis of the enzyme revealed the molecular mass of 24 kDa. The optimum pH and temperature for lipase activity were 6.5 and 37°C, respectively. The isolated lipase was stimulated by pretreatment with methanol and ethanol as well as by divalent metal ions Ca(2+), Mg(2+) and Mn(2+). The enzyme showed high activity towards oleic rich oils. The enzyme immobilized on celite could retain 90% lipase activity after eight cycles. Transesterification of Botryococcus sp. oil using the immobilized enzyme for 40 h resulted in 80% yield of fatty acid methyl esters which had good properties for use as biodiesel. Overall results suggested that the solvent tolerant Bacillus lipase can be a potential biocatalyst for methyl ester production.

  17. Simple procedure applying lactose induction and one-step purification for high-yield production of rhCIFN.

    PubMed

    Bashir, Hamid; Ahmed, Nadeem; Khan, Mohsin Ahmad; Zafar, Ahmad Usman; Tahir, Saad; Khan, Muhammad Islam; Khan, Faidad; Husnain, Tayyab

    2016-09-01

    Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5 kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl-β-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as inducer. For economical and commercial-scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14 g/L) in shake flask experiment resulted in higher yield as compared with 1 mM IPTG. Finally, with single-step purification on DEAE sepharose, 150 mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot-scale production of recombinant proteins at low cost.

  18. URANIUM RECOVERY AND PURIFICATION PROCESS AND PRODUCTION OF HIGH PURITY URANIUM TETRAFLUORIDE

    DOEpatents

    Bailes, R.H.; Long, R.S.; Grinstead, R.R.

    1957-09-17

    A process is described wherein an anionic exchange technique is employed to separate uramium from a large variety of impurities. Very efficient and economical purification of contamimated uranium can be achieved by treatment of the contaminated uranium to produce a solution containing a high concentration of chloride. Under these conditions the uranium exists as an aniomic chloride complex. Then the uranium chloride complex is adsorbed from the solution on an aniomic exchange resin, whereby a portion of the impurities remain in the solution and others are retained with the uramium by the resin. The adsorbed impurities are then removed by washing the resin with pure concentrated hydrochloric acid, after which operation the uranium is eluted with pure water yielding an acidic uranyl chloride solution of high purity.

  19. 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.

  20. Expression, Purifications, Crystallization and Preliminary X-ray Diffraction Analysis of Arabidopsis thaliana Cyclophilin 38 (AtCyp38)

    SciTech Connect

    Vasudevan,D.; Gopalon, G.; He, Z.; et. al.

    2005-01-01

    AtCyp38 is one of the highly divergent multidomain cyclophilins from Arabidopsis thaliana. A recombinant form of AtCyp38 (residues 83-437) was expressed in Escherichia coli and purified to homogeneity. The protein was crystallized using the vapour-batch technique with PEG 6000 and t-butanol as precipitants. Crystals of recombinant AtCyp38 diffracted X-rays to better than 2.5 Angstroms resolution at 95 K using a synchrotron-radiation source. The crystal belongs to the C-centered orthorhombic space group C222{sub 1}, with unit-cell parameters a = 58.2, b = 95.9, c = 167.5 Angstroms, and contains one molecule in the asymmetric unit. The selenomethionine derivative of the AtCyp38 protein was overexpressed, purified and crystallized in the same space group and data were collected to 3.5 Angstroms at the NSLS synchrotron. The structure is being solved by the MAD method.

  1. Expression, purification, crystallization and preliminary X-ray diffraction of a novel Nitrosomonas europaea cytochrome, cytochrome P460

    SciTech Connect

    Elmore, Bradley O.; Pearson, Arwen R.; Wilmot, Carrie M.; Hooper, Alan B.

    2006-04-01

    Cytochrome P460 from N. europaea, a novel mono-heme protein containing an unusual lysine cross-link to the porphyrin ring, has been recombinantly expressed and purified from E. coli and crystallized. The crystals belong to the trigonal space group P3{sub 1/2}21, with unit-cell parameters a = b = 53.3, c = 127.1 Å, one monomer in the asymmetric unit and diffract to 1.7 Å on a Cu Kα rotating-anode X-ray source. Cytochrome P460 from Nitrosomonas europaea, a novel mono-heme protein containing an unusual cross-link between a conserved lysine and the porphyrin ring, has been recombinantly expressed and purified from Escherichia coli. The protein crystallizes readily and diffraction to 1.7 Å has been obtained in-house. The crystals belong to the trigonal space group P3{sub 1/2}21, with unit-cell parameters a = b = 53.3, c = 127.1 Å, and contain one monomer in the asymmetric unit.

  2. 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.

  3. 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.

  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. Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus.

    PubMed

    Wilkens, Casper; Poulsen, Jens-Christian N; Ramløv, Hans; Lo Leggio, Leila

    2014-08-01

    Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.

  6. Purification, crystallization and initial X-ray crystallographic analysis of the putative GTPase PH0525 from Pyrococcus horikoshii OT3

    SciTech Connect

    Lokanath, Neratur K.; Yamamoto, Hitoshi; Matsunaga, Emiko; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-10-01

    The putative GTPase PH0525 from P. horikoshii OT3 was crystallized using the microbatch method. Crystals were formed under two different conditions, providing two distinct crystal forms. Diffraction data from the two forms were measured to resolution limits of 2.30 and 2.40 Å and processed in space groups P2{sub 1} and C222{sub 1}, respectively. GTPases are involved in diverse cellular functions including cell proliferation, cytoskeleton organization and intracellular traffic. The putative GTPase PH0525 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Two distinct crystal forms were grown by the microbatch method at 291 K using a very high protein concentration (80 mg ml{sup −1}). Native data sets extending to resolutions of 2.3 and 2.4 Å have been collected and processed in space groups P2{sub 1} and C222{sub 1}, respectively. Assuming the presence of one monomer per asymmetric unit gives V{sub M} values of 2.6 and 2.4 Å{sup 3} Da{sup −1} for the P2{sub 1} and C222{sub 1} forms, respectively, which is consistent with dynamic light-scattering experiments, which show a monomeric state of the protein in solution.

  7. Cloning, purification crystallization and preliminary X-ray characterization of a conserved hypothetical protein XC6422 from Xanthomonas campestris

    SciTech Connect

    Yang, Chao-Yu; Chin, Ko-Hsin; Chou, Chia-Cheng; Shr, Hui-Lin; Gao, Fei Philip; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-07-01

    A conserved hypothetical protein XC6422 from X. campestris pv. campestris has been overexpressed in E. coli, purified and crystallized. Crystals obtained from the purified recombinant protein showed a variety of forms that diffracted to at least 1.6 Å resolution. Xanthomonas campestris pv. campestris is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, roughly one third of which have no known structure and/or function. However, some genes of unknown function are highly conserved among several different bacterial genuses. XC6422 is one such conserved hypothetical protein and has been overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. Crystals grew to approximately 2 × 1.5 × 0.4 mm in size after one week and diffracted to at least 1.6 Å resolution. They belong to the monoclinic space group C2, with one molecule per asymmetric unit and unit-cell parameters a = 75.8, b = 79.3, c = 38.2 Å, β = 109.4°. Determination of this structure may provide insights into the protein’s function.

  8. Cloning, purification, crystallization and preliminary X-ray analysis of XC229, a conserved hypothetical protein from Xanthomonas campestris

    SciTech Connect

    Chin, Ko-Hsin; Kuo, Wei-Tien; Chou, Chia-Cheng; Shr, Hui-Lin; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-07-01

    A conserved hypothetical protein XC229 from X. campestris pv. campestris has been overexpressed in E. coli, purified and crystallized. A crystal of the purified recombinant protein diffracted to a resolution of 1.80 Å. Xanthomonas campestris pv. campestris is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, roughly one third of which have no known structure and/or function. However, some of these unknown genes are highly conserved among several different bacterial genuses. XC229 is one such protein containing 134 amino acids. It was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to a resolution of at least 1.80 Å. It is cubic and belongs to space group I2{sub x}3, with unit-cell parameters a = b = c = 106.8 Å. It contains one or two molecules per asymmetric unit.

  9. 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.

  10. Multifractal modeling of the production of concentrated sugar syrup crystal

    NASA Astrophysics Data System (ADS)

    Sheng, Bi; Jianbo, Gao

    2016-07-01

    High quality, concentrated sugar syrup crystal is produced in a critical step in cane sugar production: the clarification process. It is characterized by two variables: the color of the produced sugar and its clarity degree. We show that the temporal variations of these variables follow power-law distributions and can be well modeled by multiplicative cascade multifractal processes. These interesting properties suggest that the degradation in color and clarity degree has a system-wide cause. In particular, the cascade multifractal model suggests that the degradation in color and clarity degree can be equivalently accounted for by the initial “impurities” in the sugarcane. Hence, more effective cleaning of the sugarcane before the clarification stage may lead to substantial improvement in the effect of clarification.

  11. High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification.

    PubMed

    Richter, Sven; Nieveler, Jens; Schulze, Holger; Bachmann, Till T; Schmid, Rolf D

    2006-04-05

    The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.

  12. 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}.

  13. Effect of self-degradation products on crystallization of protease thermolysin

    NASA Astrophysics Data System (ADS)

    Sazaki, Gen; Aoki, Satoshi; Ooshima, Hiroshi; Kato, Jyoji

    1994-05-01

    The effect of self-degradation products of protease thermolysin on the crystallization of thermolysin was investigated. Crystallizations were carried out at the concentration of the self-degradation products of 0 to 0.622 mg/ml, 5 C, and pH 7.0. The initial concentration of thermolysin was constant (1.70 +/- 0.01 mg/ml). Crystallizations were monitored by dynamic light scattering and photomicroscopy. The crystallization of thermolysin in the presence of the self-degradation products proceeded through two successive steps: the formation of primary particles and the formation of large crystals by the aggregation of the primary particles. Low concentration of the self-degradation products (0.212 mg/ml) accelerated the formation of the primary particles and also the formation of the large crystals. High concentration of the self-degradation products, however, inhibited the formation of the primary particles and their aggregation to the large crystals. As the result, a large number of small aggregates which had not grown to the large crystals were observed by photomicroscopy. An analysis of the crystals and the primary particles formed in the presence of the self-degradation products by gel filtration high performance liquid chromatography revealed that the self-degradation products are not incorporated in the primary particles, but are incorporated probably in the openings between the primary particles during the crystallization.

  14. Automated two-column purification of iminobiotin and BrdU-labeled PCR products for rapid cloning: application to genes synthesized by polymerase chain assembly.

    PubMed

    TerMaat, Joel R; Mamedov, Tarlan G; Pienaar, Elsje; Whitney, Scott E; Subramanian, Anuradha

    2010-02-01

    Polymerase chain assembly (PCA) is a powerful tool for basic biological research and biotechnology applications. During the last several years, major advances have been made in de novo gene synthesis. However, there is still a need for fast and reproducible methods to automatically purify the synthesized genes. Upon completion of PCA, the subsequent PCR-amplified product mixture still contains undesired shorter DNA fragments that hinder cloning efforts. To avoid tedious gel purification, an automated two-column purification has been developed and used in conjunction with rapid PCA. The system enables fast synthesis and isolation of the full-length DNA of interest, important for facile cloning of desired DNA fragments. During the PCR amplification step, forward and reverse primers tagged with iminobiotin and bromodeoxyuridine labels, respectively, were used. The automated purification was then performed on the PCR mixture using two affinity/immunocapture columns in series to isolate only the desired full-length product. The procedure has been applied to the pUC19 beta-lactamase gene (929 bp). Follow-up PCR of the purified product, cloning, and sequencing demonstrated the technique's effectiveness in obtaining the pure full-length gene. The purification has also been performed on other synthesized genes, indicating its utility as a general approach.

  15. Cosmogenic radionuclide production in NaI(Tl) crystals

    NASA Astrophysics Data System (ADS)

    Amaré, J.; Cebrián, S.; Cuesta, C.; García, E.; Ginestra, C.; Martínez, M.; Oliván, M. A.; Ortigoza, Y.; Ortiz de Solórzano, A.; Pobes, C.; Puimedón, J.; Sarsa, M. L.; Villar, J. A.; Villar, P.

    2015-02-01

    The production of long-lived radioactive isotopes in materials due to the exposure to cosmic rays on Earth surface can be an hazard for experiments demanding ultra-low background conditions, typically performed deep underground. Production rates of cosmogenic isotopes in all the materials present in the experimental set-up, as well as the corresponding cosmic rays exposure history, must be both well known in order to assess the relevance of this effect in the achievable sensitivity of a given experiment. Although NaI(Tl) scintillators are being used in experiments aiming at the direct detection of dark matter since the first nineties of the last century, very few data about cosmogenic isotopes production rates have been published up to date. In this work we present data from two 12.5 kg NaI(Tl) detectors, developed in the frame of the ANAIS project, which were installed inside a convenient shielding at the Canfranc Underground Laboratory just after finishing surface exposure to cosmic rays. The very fast start of data taking allowed to identify and quantify isotopes with half-lives of the order of tens of days. Initial activities underground have been measured and then production rates at sea level have been estimated following the history of detectors; values of about a few tens of nuclei per kg and day for Te isotopes and 22Na and of a few hundreds for I isotopes have been found. These are the first direct estimates of production rates of cosmogenic nuclides in NaI crystals. A comparison of the so deduced rates with calculations using typical cosmic neutron flux at sea level and a carefully selected description of excitation functions will be also presented together with an estimate of the corresponding contribution to the background at low and high energies, which can be relevant for experiments aiming at rare events searches.

  16. 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

  17. Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 7

    SciTech Connect

    Quesada, Odayme; Gurda, Brittney; Govindasamy, Lakshmanan; McKenna, Robert; Kohlbrenner, Erik; Aslanidi, George; Zolotukhin, Sergei; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2007-12-01

    Crystals of baculovirus-expressed adeno-associated virus serotype 7 capsids have been produced which diffract X-rays to ∼3.0 Å resolution. Crystals of baculovirus-expressed adeno-associated virus serotype 7 capsids diffract X-rays to ∼3.0 Å resolution. The crystals belong to the rhombohedral space group R3, with unit-cell parameters a = 252.4, c = 591.2 Å in the hexagonal setting. The diffraction data were processed and reduced to an overall completeness of 79.0% and an R{sub merge} of 12.0%. There are three viral capsids in the unit cell. The icosahedral threefold axis is coincident with the crystallographic threefold axis, resulting in one third of a capsid (20 monomers) per crystallographic asymmetric unit. The orientation of the viral capsid has been determined by rotation-function searches and is positioned at (0, 0, 0) by packing considerations.

  18. Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag

    PubMed Central

    Jayanthi, Srinivas; Koppolu, Bhanu prasanth; Smith, Sean G.; Jalah, Rashmi; Bear, Jenifer; Rosati, Margherita; Pavlakis, George N.; Felber, Barbara K.; Zaharoff, David A.; Kumar, Thallapuranam Krishnaswamy Suresh

    2014-01-01

    Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous.Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ~98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichrosim, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31 ± 0.05 mg/ mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app) = 69 ± 1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics. PMID:25123642

  19. Purification, crystallization and preliminary X-ray analysis of a fusion of the LIM domains of LMO2 and the LID domain of Ldb1.

    PubMed

    El Omari, Kamel; Porcher, Catherine; Mancini, Erika J

    2010-11-01

    LMO2 (LIM domain only 2), also known as rhombotin-2, is a transcriptional regulator that is essential for normal haematopoietic development. In malignant haematopoiesis, its ectopic expression in T cells is involved in the pathogenesis of leukaemia. LMO2 contains four zinc-finger domains and binds to the ubiquitous nuclear adaptor protein Ldb1 via the LIM-interaction domain (LID). Together, they act as scaffolding proteins and bridge important haematopoietic transcription factors such as SCL/Tal1, E2A and GATA-1. Solving the structure of the LMO2:Ldb1-LID complex would therefore be a first step towards understanding how haematopoietic specific protein complexes form and would also provide an attractive target for drug development in anticancer therapy, especially for T-cell leukaemia. Here, the expression, purification, crystallization and data collection of a fusion protein consisting of the two LIM domains of LMO2 linked to the LID domain of Ldb1 via a flexible linker is reported. The crystals belonged to space group C2, with unit-cell parameters a = 179.9, b = 51.5, c = 114.7 Å, β = 90.1°, and contained five molecules in the asymmetric unit. Multiple-wavelength anomalous dispersion (MAD) data have been collected at the zinc X-ray absorption edge to a resolution of 2.8 Å and the data were used to solve the structure of the LMO2:Ldb1-LID complex. Refinement and analysis of the electron-density map is in progress.

  20. Expression, purification, crystallization and preliminary crystallographic study of FtsA from methicillin-resistant Staphylococcus aureus

    PubMed Central

    Fujita, Junso; Miyazaki, Yuma; Hirose, Mika; Nagao, Chioko; Mizohata, Eiichi; Matsumoto, Yoshimi; Mizuguchi, Kenji; Inoue, Tsuyoshi; Matsumura, Hiroyoshi

    2013-01-01

    FtsA from methicillin-resistant Staphylococcus aureus (MRSA) was cloned, overexpressed and purified. The protein was crystallized using the sitting-drop vapour-diffusion technique. A cocrystal with β-γ-imidoadenosine 5′-phosphate (AMPPNP; a nonhydrolysable ATP analogue) was grown using PEG 3350 as a precipitant at 293 K. X-ray diffraction data were collected to a resolution of 2.3 Å at 100 K. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 75.31, b = 102.78, c = 105.90 Å, β = 96.54°. The calculated Matthews coefficient suggested that the asymmetric unit contained three or four monomers. PMID:23908037

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

    PubMed

    Han, Huijong; Kursula, Petri

    2014-11-01

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

  2. Purification, crystallization and preliminary X-ray diffraction studies on human Ca2+-binding protein S100B.

    PubMed

    Ostendorp, Thorsten; Heizmann, Claus W; Kroneck, Peter M H; Fritz, Günter

    2005-07-01

    S100B, a Ca2+-binding protein, acts intracellularly as a Ca2+-signalling protein but is also secreted to the extracellular space, acting in a cytokine-like manner through its receptor RAGE. Recombinant human S100B has been purified and crystallized in the Ca2+-bound state. Size-exclusion chromatography indicates that S100B can exist as a dimer and as a multimer in solution. Crystals of S100B diffract to 1.9 A and belong to space group P2(1), with unit-cell parameters a = 63.4, b = 81.6, c = 71.5 A, alpha = 90, beta = 107, gamma = 90 degrees. Preliminary analysis of the X-ray data indicate that there are four homodimers per asymmetric unit.

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

    PubMed Central

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

    2009-01-01

    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 C2221, with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 Å. Diffraction data were collected to a resolution of 2.7 Å. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%. PMID:19574654

  4. Cloning, expression, purification, crystallization and preliminary X-ray analysis of EaLsc, a levansucrase from Erwinia amylovora.

    PubMed

    Caputi, Lorenzo; Cianci, Michele; Benini, Stefano

    2013-05-01

    The Gram-negative bacterium Erwinia amylovora is a destructive pathogen of Rosaceae. During infection, E. amylovora produces the exopolysaccharide levan, which contributes to the occlusion of plant vessels, causing the wilting of shoots. Levan is a fructose polymer that is synthesized by multifunctional enzymes called levansucrases. The levansucrase from E. amylovora (EaLsc) was heterologously expressed as a GST-fusion protein in Escherichia coli, purified and crystallized after tag removal. The protein crystallized in space group P21212. X-ray diffraction data were acquired to 2.77 Å resolution. The structure of the enzyme was solved by molecular replacement. The asymmetric unit contains eight enzyme molecules, giving a solvent content of 58.74% and a Matthews coefficient of 2.98 Å(3) Da(-1).

  5. 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}.

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

    PubMed Central

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

    2014-01-01

    Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1—C2 amide bond of the five-membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C-terminal His6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 M potassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Å with an R merge of 29.2% from a crystal belonging to space group P1211, with unit-cell parameters a = 54.93, b = 164.74, c = 106.89 Å, β = 98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (V M = 2.34 Å3 Da−1). PMID:25372819

  7. Expression, purification and crystallization of a membrane-associated, catalytically active type I signal peptidase from Staphylococcus aureus.

    PubMed

    Ting, Yi Tian; Batot, Gaëlle; Baker, Edward N; Young, Paul G

    2015-01-01

    Staphylococcus aureus infections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets. S. aureus has a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, the spsB gene from S. aureus Newman strain was cloned and overexpressed in Escherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space group P2(1) and diffracted to 2.05 Å resolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases.

  8. 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)).

  9. 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.

  10. Purification, crystallization and preliminary crystallographic analysis of DehIVa, a dehalogenase from Burkholderia cepacia MBA4.

    PubMed

    Schmidberger, Jason W; Oakley, Aaron J; Tsang, Jimmy S H; Wilce, Matthew C J

    2005-03-01

    DehIVa is one of two dehalogenases produced by the soil- and water-borne bacterium Burkholderia cepacia MBA4. It acts to break down short-chain halogenated aliphatic acids through a nucleophilic attack and subsequent hydrolysis of an enzyme-substrate intermediate to remove the halide ions from L-enantiomers substituted at the C2 position (e.g L-2-monochloropropionic acid). Dehalogenases are an important group of enzymes that are responsible for breaking down a diverse range of halogenated environmental pollutants. The dhlIVa gene coding for DehIVa was expressed in Escherichia coli and the protein was purified and crystallized using the hanging-drop method. Crystals grown in PEG 4000 and ammonium sulfate diffracted to 3.1 A. The crystals had a primitive hexagonal unit cell, with unit-cell parameters a = b = 104.2, c = 135.8 A, alpha = beta = 90, gamma = 120 degrees. Determining this structure will provide valuable insights into the characterization of the catalytic mechanisms of this group of enzymes.

  11. 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.

  12. 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.

  13. Expression, purification, crystallization and preliminary X-ray characterization of the GRP carbohydrate-recognition domain from Homo sapiens

    SciTech Connect

    Zhou, Dongwen; Sun, Jianping; Zhao, Wei; Zhang, Xiao; Shi, Yunyu; Teng, Maikun Niu, Liwen; Dong, Yuhui; Liu, Peng

    2006-05-01

    The CRD domain of GRP from H. sapiens has been expressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.0 Å. Galectins are a family of animal lectins which share similar carbohydrate-recognition domains (CRDs) and an affinity for β-galactosides. A novel human galectin-related protein named GRP (galectin-related protein; previously known as HSPC159) comprises only one conserved CRD with 38 additional N-terminal residues. The C-terminal fragment of human GRP (GRP-C; residues 38–172) containing the CRD has been expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 2% PEG 400 and 2M ammonium sulfate in 100 mM Tris–HCl buffer pH 7.5. Diffraction data were collected to a resolution limit of 2.0 Å at beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 123.07, b = 96.67, c = 61.56 Å, β = 118.72°. The estimated Matthews coefficient was 2.6 Å{sup 3} Da{sup −1}, corresponding to 51.8% solvent content.

  14. 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.

  15. Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45.

    PubMed Central

    Paustian, T D; Shah, V K; Roberts, G P

    1989-01-01

    The nifN and -E gene products are involved in the synthesis of the iron-molybdenum cofactor of dinitrogenase, the enzyme responsible for the reduction of dinitrogen to ammonia. By using the in vitro iron-molybdenum cofactor biosynthesis assay, we have followed the purification of these gene products 450-fold to greater than 95% purity. An overall recovery of 20% was obtained with the purified protein having a specific activity of 6900 units/mg of protein. The protein (hereafter referred to as NIFNE) was found to contain equimolar amounts of the nifN and -E gene products and have a native molecular mass of 200 +/- 10 kDa, which indicates an alpha 2 beta 2 structure. NIFNE was oxygen labile with a half-life of 1 min in air. A UV-visible spectrum of the dye-oxidized protein showed an absorption maximum at 425 nm that could be bleached by reduction of NIFNE with sodium dithionite, suggesting the presence of an Fe center in NIFNE. Images PMID:2668954

  16. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the novel modular DNA-binding protein BurrH in its apo form and in complex with its target DNA.

    PubMed

    Stella, Stefano; Molina, Rafael; Bertonatti, Claudia; Juillerrat, Alexandre; Montoya, Guillermo

    2014-01-01

    Different genome-editing strategies have fuelled the development of new DNA-targeting molecular tools allowing precise gene modifications. Here, the expression, purification, crystallization and preliminary X-ray diffraction of BurrH, a novel DNA-binding protein from Burkholderia rhizoxinica, are reported. Crystallization experiments of BurrH in its apo form and in complex with its target DNA yielded crystals suitable for X-ray diffraction analysis. The crystals of the apo form belonged to the primitive hexagonal space group P3(1) or its enantiomorph P3(2), with unit-cell parameters a = b = 73.28, c = 268.02 Å, α = β = 90, γ = 120°. The BurrH-DNA complex crystallized in the monoclinic space group P2(1), with unit-cell parameters a = 70.15, b = 95.83, c = 76.41 Å, α = γ = 90, β = 109.51°. The self-rotation function and the Matthews coefficient suggested the presence of two protein molecules per asymmetric unit in the apo crystals and one protein-DNA complex in the monoclinic crystals. The crystals diffracted to resolution limits of 2.21 and 2.65 Å, respectively, using synchrotron radiation.

  17. Glycyl endopeptidase from papaya latex: partial purification and use for production of fish gelatin hydrolysate.

    PubMed

    Karnjanapratum, Supatra; Benjakul, Soottawat

    2014-12-15

    An aqueous two-phase system (ATPS) in combination with ammonium sulphate ((NH4)2SO4) precipitation was applied to fractionate glycyl endopeptidase from the papaya latex of Red Lady and Khack Dum cultivars. ATPS containing polyethylene glycol (PEG 2000 and 6000) and salts ((NH4)2SO4 and MgSO4) at different concentrations were used. Glycyl endopeptidase with high purification fold (PF) and yield was found in the salt-rich bottom phase of ATPS with 10%PEG 6000-10% (NH4)2SO4. When ATPS fraction from Red Lady cultivar was further precipitated with 40-60% saturation of (NH4)2SO4, PF of 2.1-fold with 80.23% yield was obtained. Almost all offensive odorous compounds, particularly benzyl isothiocyanate, were removed from partially purified glycyl endopeptidase (PPGE). The fish gelatin hydrolysates prepared using PPGE showed higher ABTS radical scavenging activity and less odour, compared with those of crude extract (CE). Thus antioxidative gelatin hydrolysate with negligible undesirable odour could be prepared with the aid of PPGE.

  18. Monoclonal antibodies to human interferon-gamma: production, affinity purification and radioimmunoassay.

    PubMed Central

    Novick, D; Eshhar, Z; Fischer, D G; Friedlander, J; Rubinstein, M

    1983-01-01

    Human interferon-gamma (IFN-gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN-gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN-gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN-gamma. Moreover, IFN-gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN-gamma subtypes are immunologically cross-reactive. Immune precipitation of partially purified 125I-labelled IFN-gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN-gamma gave a 50-fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid-phase radioimmunoassay, allowing the detection of IFN-gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h. Images Fig. 1. Fig. 2. PMID:11892806

  19. Expression, purification, crystallization and preliminary X-ray characterization of the GRP carbohydrate-recognition domain from Homo sapiens

    PubMed Central

    Zhou, Dongwen; Sun, Jianping; Zhao, Wei; Zhang, Xiao; Shi, Yunyu; Teng, Maikun; Niu, Liwen; Dong, Yuhui; Liu, Peng

    2006-01-01

    Galectins are a family of animal lectins which share similar carbohydrate-recognition domains (CRDs) and an affinity for β-galactosides. A novel human galectin-related protein named GRP (galectin-related protein; previously known as HSPC159) comprises only one conserved CRD with 38 additional N-terminal residues. The C-terminal fragment of human GRP (GRP-C; residues 38–172) containing the CRD has been expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 2% PEG 400 and 2M ammonium sulfate in 100 mM Tris–HCl buffer pH 7.5. Diffraction data were collected to a resolution limit of 2.0 Å at beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 123.07, b = 96.67, c = 61.56 Å, β = 118.72°. The estimated Matthews coefficient was 2.6 Å3 Da−1, corresponding to 51.8% solvent content. PMID:16682780

  20. Purification and characterization of a rabbit salivary protein, a potent inhibitor of crystal growth of calcium phosphate salts.

    PubMed

    Spielman, A I; Bernstein, A; Hay, D I; Blum, M; Bennick, A

    1991-01-01

    Human saliva is supersaturated with respect to basic calcium phosphate salts but is stabilized by specific macromolecules that inhibit calcium phosphate precipitation. One of the families of inhibitory proteins in human and monkey saliva is the acidic proline-rich proteins. The purpose of this study was to isolate and characterize inhibitors of calcium phosphate precipitation from rabbit parotid saliva. Saliva was fractionated by immunoaffinity chromatography and anion exchange chromatography. Individual fractions were assayed for their ability to inhibit calcium phosphate crystal growth and the fraction associated with the inhibition was purified by repeated anion exchange chromatography, preparative gel electrophoresis and electroelution. A major (APRP) and two minor proteins (AM1, AM2) that were inhibitory were purified. APRP is an acidic proline-rich phospho-glycoprotein and a very potent inhibitor of secondary crystal growth of calcium phosphate as it was active at a concentration of 2 x 10(-8) M in a standard assay. The N-terminal sequence of one APRP was EYENLDGSLAATQNDDD?Q and a clostripain fragment of APRP had the following N-terminal sequence PQHRPPRPGGH-????SPPP?GN???PPP. Although the N-terminal segment of APRP does not resemble that of proline-rich proteins, alignment of the clostripain fragment with the repeat region of such proteins from rat, mouse, monkey and man revealed a high degree of similarity, indicating a structural relationship with the proline-rich protein family.

  1. 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.

  2. Purification, crystallization and preliminary crystallographic analysis of the globular domain of the human type V myosin Myo5a.

    PubMed

    Velvarska, Hana; Niessing, Dierk

    2013-11-01

    Type V myosins constitute the main cargo-transporting class of myosin motors in higher eukaryotes. They are mainly defined by their C-terminal globular domain, which is required for cargo binding as well as for motor auto-inhibition in the absence of cargo. To date, high-resolution structures only exist for globular domains from yeast. Since the majority of cellular cargoes in yeast are very different from the cargoes in higher eukaryotes, structural insights into the domain organization of globular domains from human type V myosins are important. The globular domain of human Myo5a was cloned, expressed and crystallized and data sets were collected. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 75.04, b = 86.70, c = 131.41 Å, α = β = γ = 90°, and diffracted with data-collection quality to 2.5 Å resolution.

  3. Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain.

    PubMed

    Agarwal, Rakhi; Eswaramoorthy, Subramaniam; Kumaran, Desigan; Dunn, John J; Swaminathan, Subramanyam

    2004-03-01

    The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1A.

  4. 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.

  5. Improved expression, purification and crystallization of a putative N-acetyl-γ-glutamyl-phosphate reductase from rice (Oryza sativa)

    SciTech Connect

    Miura-Ohnuma, Jun; Nonaka, Tsuyoshi; Katoh, Shizue; Murata, Katsuyoshi; Kita, Akiko; Miki, Kunio

    2005-12-01

    Crystals of OsAGPR were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P6{sub 1}, with unit-cell parameters a = 86.11, c = 316.3 Å. N-Acetyl-γ-glutamyl-phosphate reductase (AGPR) catalyzes the third step in an eight-step arginine-biosynthetic pathway that starts with glutamate. This enzyme converts N-acetyl-γ-glutamyl phosphate to N-acetylglutamate-γ-semialdehyde by an NADPH-dependent reductive dephosphorylation. AGPR from Oryza sativa (OsAGPR) was expressed in Escherichia coli at 291 K as a soluble fusion protein with an upstream thioredoxin-hexahistidine [Trx-(His){sub 6}] extension. OsAGPR(Ala50–Pro366) was purified and crystals were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P6{sub 1}, with unit-cell parameters a = 86.11, c = 316.3 Å.

  6. 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.

  7. Determination of Vitamin B12 in meat products by RP-HPLC after enrichment and purification on an immunoaffinity column.

    PubMed

    Guggisberg, D; Risse, M C; Hadorn, R

    2012-02-01

    A quantitative method for the determination of Vitamin B12 in meat products by RP-HPLC and UV detection was developed and compared to the reference method (microbiological assay, MBA). Vitamin B12 was extracted with 50mM sodium acetate buffer in the presence of sodium cyanide. For the quantification of total Vitamin B12, it was necessary to release protein-bound Vitamin B12 by pepsin treatment. Cyanocobalamin was detected as total Vitamin B12 after purification and enrichment on an immunoaffinity column. The calibration with five concentrations of Vitamin B12 was linear with a regression coefficient r2>0.99. The method was validated at three different concentration levels (5-15 ng/g) with salami showing good recovery rates between 80 and 108% and low relative standard deviations between 1.50 and 7.26% (n = 6). The detection limit was found to be 2 ng/g. The Vitamin B12 levels of 50 meat products measured by the developed procedure were similar or significantly lower than those determined by the MBA.

  8. Demonstration of a strategy for product purification by high-gradient magnetic fishing: recovery of superoxide dismutase from unconditioned whey.

    PubMed

    Meyer, Andrea; Hansen, Dennis B; Gomes, Cláudia S G; Hobley, Timothy J; Thomas, Owen R T; Franzreb, Matthias

    2005-01-01

    A systematic approach for the design of a bioproduct recovery process employing magnetic supports and the technique of high-gradient magnetic fishing (HGMF) is described. The approach is illustrated for the separation of superoxide dismutase (SOD), an antioxidant protein present in low concentrations (ca. 0.15-0.6 mg L(-1)) in whey. The first part of the process design consisted of ligand screening in which metal chelate supports charged with copper(II) ions were found to be the most suitable. The second stage involved systematic and sequential optimization of conditions for the following steps: product adsorption, support washing, and product elution. Next, the capacity of a novel high-gradient magnetic separator (designed for biotechnological applications) for trapping and holding magnetic supports was determined. Finally, all of the above elements were assembled to deliver a HGMF process for the isolation of SOD from crude sweet whey, which consisted of (i) binding SOD using Cu2+ -charged magnetic metal chelator particles in a batch reactor with whey; (ii) recovery of the "SOD-loaded" supports by high-gradient magnetic separation (HGMS); (iii) washing out loosely bound and entrained proteins and solids; (iv) elution of the target protein; and (v) recovery of the eluted supports from the HGMF rig. Efficient recovery of SOD was demonstrated at approximately 50-fold increased scale (cf magnetic rack studies) in three separate HGMF experiments, and in the best of these (run 3) an SOD yield of >85% and purification factor of approximately 21 were obtained.

  9. Purification, crystallization and preliminary X-ray diffraction analysis of the Staphylococcus epidermidis extracellular serine protease Esp

    PubMed Central

    Vengadesan, Krishnan; Macon, Kevin; Sugumoto, Shinya; Mizunoe, Yoshimitsu; Iwase, Tadayuki; Narayana, Sthanam V. L.

    2013-01-01

    Esp, an extracellular serine protease from Staphylococcus epidermidis, has been shown to inhibit S. aureus biofilm formation and nasal colonization. The full-length 27 kDa pro-Esp was purified and digested with thermolysin to obtain mature Esp. The mature Esp containing 216 residues crystallized in space group P21, with unit-cell parameters a = 39.5, b = 61.2, c = 42.5 Å, β = 98.2° and one molecule in the asymmetric unit, with an estimated solvent content of 42%. A diffraction data set has been collected to 1.8 Å resolution on a rotating-anode home-source facility. PMID:23295486

  10. Purification, crystallization and preliminary X-ray diffraction analysis of the Staphylococcus epidermidis extracellular serine protease Esp.

    PubMed

    Vengadesan, Krishnan; Macon, Kevin; Sugumoto, Shinya; Mizunoe, Yoshimitsu; Iwase, Tadayuki; Narayana, Sthanam V L

    2013-01-01

    Esp, an extracellular serine protease from Staphylococcus epidermidis, has been shown to inhibit S. aureus biofilm formation and nasal colonization. The full-length 27 kDa pro-Esp was purified and digested with thermolysin to obtain mature Esp. The mature Esp containing 216 residues crystallized in space group P2(1), with unit-cell parameters a = 39.5, b = 61.2, c = 42.5 Å, β = 98.2° and one molecule in the asymmetric unit, with an estimated solvent content of 42%. A diffraction data set has been collected to 1.8 Å resolution on a rotating-anode home-source facility.

  11. Agarose gel purification of PCR products for denaturing gradient gel electrophoresis results in GC-clamp deletion.

    PubMed

    Sun, Guowei; Xiao, Jinzhou; Lu, Man; Wang, Hongming; Chen, Xiaobing; Yu, Yongxin; Pan, Yingjie; Wang, Yongjie

    2015-01-01

    The 16S ribosomal RNA (rRNA) gene of marine archaeal samples was amplified using a nested PCR approach, and the V3 region of 16S rRNA gene of crab gut microbiota (CGM) was amplified using the V3 universal primer pair with a guanine and cytosine (GC)-clamp. Unpurified PCR products (UPPs), products purified from reaction solution (PPFSs), and products purified from gel (PPFGs) of above two DNA samples were used for denaturing gradient gel electrophoresis (DGGE) analysis, respectively. In contrast to almost identical band patterns shared by both the UPP and PPFS, the PPFGs were barely observed on the DGGE gel for both the marine archaea and CGM samples. Both PPFS and PPFG of CGM V3 regions were subjected to cloning. A small amount of positive clones was obtained for PPFS, but no positive clones were observed for PPFG. The melt curve and direct sequencing analysis of PPFS and PPFG of E. coli V3 region indicated that the Tm value of PPFG (82.35 ± 0.19 °C) was less than that of PPFS (83.81 ± 0.11 °C), and the number of shorter GC-clamps was significant higher in PPFG than in PPFS. The ultraviolet exposure experiment indicated that the ultraviolet was not responsible for the deletion of the GC-clamps. We conclude that the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples.

  12. PURIFICATION OF PLUTONIUM USING A CERIUM PRECIPITATE AS A CARRIER FOR FISSION PRODUCTS

    DOEpatents

    Faris, B.F.; Olson, C.M.

    1961-07-01

    Bismuth phosphate carrier precipitation processes are described for the separation of plutonium from fission products wherein in at least one step bismuth phosphate is precipitated in the presence of hexavalent plutonium thereby carrying a portion of the fission products from soluble plu tonium values. In this step, a cerium phosphate precipitate is formed in conjunction with the bismuth phosphate precipitate, thereby increasing the amount of fission products removed from solution.

  13. Production and purification of high-titer foamy virus vector for the treatment of leukocyte adhesion deficiency

    PubMed Central

    Nasimuzzaman, Md; Lynn, Danielle; Ernst, Rebecca; Beuerlein, Michele; Smith, Richard H.; Shrestha, Archana; Cross, Scott; Link, Kevin; Lutzko, Carolyn; Nordling, Diana; Russell, David W.; Larochelle, Andre; Malik, Punam; Van der Loo, Johannes C.M.

    2016-01-01

    Compared to other integrating viral vectors, foamy virus (FV) vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34+ cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy. However, infectious titers of FV-human(h)CD18 were low and not suitable for manufacturing of clinical-grade product. Herein, we developed a scalable production and purification process that resulted in 60-fold higher FV-hCD18 titers from ~1.7 × 104 to 1.0 × 106 infectious units (IU)/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimized gag, heparin affinity chromatography, tangential flow filtration, and ultracentrifugation, which reproducibly resulted in 5,000-fold concentrated and purified virus, an overall yield of 19 ± 3%, and final titers of 1–2 × 109 IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO) concentration, thereby avoiding DMSO-induced toxicity to CD34+ cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production. PMID:27722179

  14. 75 FR 10311 - In the Matter of Certain Liquid Crystal Display Devices and Products Containing the Same; Notice...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-05

    ... Liquid Crystal Display Devices and Products Containing the Same; Notice of Commission Determination To... importation of certain liquid crystal display (``LCD'') devices and products containing the same by reason...

  15. 75 FR 9928 - In the Matter of: Certain Liquid Crystal Display Modules, Products Containing Same, and Methods...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-04

    ... From the Federal Register Online via the Government Publishing Office ] INTERNATIONAL TRADE COMMISSION In the Matter of: Certain Liquid Crystal Display Modules, Products Containing Same, and Methods... within the United States after importation of certain liquid crystal display devices, products...

  16. Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses.

    PubMed

    Ungerechts, Guy; Bossow, Sascha; Leuchs, Barbara; Holm, Per S; Rommelaere, Jean; Coffey, Matt; Coffin, Rob; Bell, John; Nettelbeck, Dirk M

    2016-01-01

    Oncolytic viruses (OVs) are unique anticancer agents based on their pleotropic modes of action, which include, besides viral tumor cell lysis, activation of antitumor immunity. A panel of diverse viruses, often genetically engineered, has advanced to clinical investigation, including phase 3 studies. This diversity of virotherapeutics not only offers interesting opportunities for the implementation of different therapeutic regimens but also poses challenges for clinical translation. Thus, manufacturing processes and regulatory approval paths need to be established for each OV individually. This review provides an overview of clinical-grade manufacturing procedures for OVs using six virus families as examples, and key challenges are discussed individually. For example, different virus features with respect to particle size, presence/absence of an envelope, and host species imply specific requirements for measures to ensure sterility, for handling, and for determination of appropriate animal models for toxicity testing, respectively. On the other hand, optimization of serum-free culture conditions, increasing virus yields, development of scalable purification strategies, and formulations guaranteeing long-term stability are challenges common to several if not all OVs. In light of the recent marketing approval of the first OV in the Western world, strategies for further upscaling OV manufacturing and optimizing product characterization will receive increasing attention.

  17. Application of NASA's Advanced Life Support Technologies for Waste Treatment, Water Purification and Recycle, and Food Production in Polar Regions

    NASA Technical Reports Server (NTRS)

    Bubenheim, David L.; Lewis, Carol E.; Covington, M. Alan (Technical Monitor)

    1995-01-01

    NASA's advanced life support technologies are being combined with Arctic science and engineering knowledge to address the unique needs of the remote communities of Alaska through the Advanced Life Systems for Extreme Environments (ALSEE) project. ALSEE is a collaborative effort involving NASA, the State of Alaska, the University of Alaska, the North Slope Borough of Alaska, and the National Science Foundation (NSF). The focus is a major issue in the state of Alaska and other areas of the Circumpolar North, the health and welfare of its people, their lives and the subsistence lifestyle in remote communities, economic opportunity, and care for the environment. The project primarily provides treatment and reduction of waste, purification and recycling of water. and production of food. A testbed is being established to demonstrate the technologies which will enable safe, healthy, and autonomous function of remote communities and to establish the base for commercial development of the resulting technology into new industries. The challenge is to implement the technological capabilities in a manner compatible with the social and economic structures of the native communities, the state, and the commercial sector. Additional information is contained in the original extended abstract.

  18. Purification, characterization and end product analysis of dextran degrading endodextranase from Bacillus licheniformis KIBGE-IB25.

    PubMed

    Zohra, Rashida Rahmat; Aman, Afsheen; Ansari, Asma; Haider, Muhammad Samee; Qader, Shah Ali Ul

    2015-07-01

    Degradation of high molecular weight dextran for obtaining low molecular weight dextran is based on the hydrolysis using chemical and enzymatic methods. Current research study focused on production, purification and characterization of dextranase from a newly isolated strain of Bacillus licheniformis KIBGE-IB25. Dextranase was purified up to 36 folds with specific activity of 1405 U/mg and molecular weight of 158 kDa. It was found that enzyme performs optimum cleavage of dextran (5000 Da, 0.5%) at 35 °C in 15 min at pH 4.5 with a Km and Vmax of 0.374 mg/ml and 182 μmol/min, respectively. Relative amino acid composition analysis of purified enzyme suggested the presence of higher number of hydrophobic, acidic and glycosylation promoting amino acids. The N-terminal sequence of dextranase KIBGE-IB25 was AYTVTLYLQG. It exhibited distinct amino acid sequence yet shared some inherent characteristics with glycosyl hydrolases (GH) family 49 and also testified the presence of O-glycosylation at N-terminal end.

  19. Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses

    PubMed Central

    Ungerechts, Guy; Bossow, Sascha; Leuchs, Barbara; Holm, Per S; Rommelaere, Jean; Coffey, Matt; Coffin, Rob; Bell, John; Nettelbeck, Dirk M

    2016-01-01

    Oncolytic viruses (OVs) are unique anticancer agents based on their pleotropic modes of action, which include, besides viral tumor cell lysis, activation of antitumor immunity. A panel of diverse viruses, often genetically engineered, has advanced to clinical investigation, including phase 3 studies. This diversity of virotherapeutics not only offers interesting opportunities for the implementation of different therapeutic regimens but also poses challenges for clinical translation. Thus, manufacturing processes and regulatory approval paths need to be established for each OV individually. This review provides an overview of clinical-grade manufacturing procedures for OVs using six virus families as examples, and key challenges are discussed individually. For example, different virus features with respect to particle size, presence/absence of an envelope, and host species imply specific requirements for measures to ensure sterility, for handling, and for determination of appropriate animal models for toxicity testing, respectively. On the other hand, optimization of serum-free culture conditions, increasing virus yields, development of scalable purification strategies, and formulations guaranteeing long-term stability are challenges common to several if not all OVs. In light of the recent marketing approval of the first OV in the Western world, strategies for further upscaling OV manufacturing and optimizing product characterization will receive increasing attention. PMID:27088104

  20. Production, purification and characterization of halophilic organic solvent tolerant protease from marine crustacean shell wastes and its efficacy on deproteinization.

    PubMed

    Maruthiah, Thirumalai; Somanath, Beena; Jasmin, Jebamonydhas Vijila; Immanuel, Grasian; Palavesam, Arunachalam

    2016-12-01

    The quantum of marine fish wastes produced by fish processing industries has necessitated to search new methods for its disposal. Hence, this study is focused on production and purification of halophilic organic solvent tolerant protease (HOSP) from marine Alcaligenes faecalis APCMST-MKW6 using marine shell wastes as substrate. The candidate bacterium was isolated from the marine sediment of Manakudi coast and identified as A. faecalis APCMST-MKW6. The purified protease showed 16.39-fold purity, 70.34 U/mg specific activity with 21.67 % yield. The molecular weight of the purified alkaline protease was 49 kDa. This purified protease registered maximum activity at pH 9 and it was stable between pH 8-9 after 1.30 h of incubation. The optimum temperature registered was 60 °C and it was stable between 50 and 60 °C even after 1.30 h of incubation. This enzyme also showed maximum activity at 20 % NaCl concentration. Further, manganese chloride, magnesium chloride, calcium chloride and barium chloride influenced this enzyme activity remarkably and it was also found to be enhanced by many of the tested surfactants and solvents. The candidate bacterium effectively deproteinized the shrimp shell waste compared to the other tested crustaceans shell wastes and also attained maximum antioxidant activity.

  1. Lurgi's MPG gasification plus Rectisol{reg_sign} gas purification - advanced process combination for reliable syngas production

    SciTech Connect

    2005-07-01

    Lurgi's Multi Purpose Gasification Process (MPG) is the reliable partial oxidation process to convert hydrocarbon liquids, slurries and natural gas into valuable syngas. The MPG burner has once again proven its capabilities in an ammonia plant based on asphalt gasification. Lurgi is operating the HP-POX demonstration plant together with the University of Freiberg, Germany. Gasification tests at pressures of up to 100 bar have shown that syngas for high pressure synthesis such as methanol and ammonia can be produced more economically. The Rectisol{reg_sign} gas purification process yields ultra clean synthesis gas which is required to avoid problems in the downstream synthesis. Pure carbon dioxide is produced as a separate stream and is readily available for sequestration, enhanced oil recovery or other uses. The reliability of the Rectisol{reg_sign} process and the confidence of plant operators in this process are acknowledged by the fact that more than 75% of the syngas produced world wide by coal, oil and waste gasification is purified in Rectisol{reg_sign} units. Virtually all coal gasification plants currently under construction rely on Rectisol{reg_sign}. The new, large GTL plants and hydrogen production facilities require effective CO{sub 2} removal. New developments make Rectisol{reg_sign} attractive for this task. 10 figs., 3 tabs., 2 photos.

  2. Purification of liquid products of cotton wipes biotransformation with the aid of Trichoderma viridae in orbital flight

    NASA Astrophysics Data System (ADS)

    Viacheslav, Ilyin; Korshunov, Denis

    Recovery of various organic wastes in space flight is an actual problem of modern astronautics and future interplanetary missions. Currently, organic waste are incinerated in the dense layers of the Earth's atmosphere in cargo containers. However, this method of anthropogenic waste treatment is not environmentally compatible with future interplanetary missions, and is not suitable due to planetary quarantine requirements. Furthermore, the maintaining of a closed ecosystem in spaceship is considered as one of the main ways of ensuring the food and air crew in the long term fully autonomous space expedition. Such isolated ecosystem is not conceivable without biotransformation of organic waste. In this regard, currently new ways of recycling organic waste are currently developed. The most promising method is a method for processing organic waste using thermophilic anaerobic microbial communities.However, the products of anaerobic fermentation of solid organic materials contain significant amounts of organic impurities, which often give them sour pH. This presents a significant problem because it does not allow to use this fluid as process water without pretreatment. Fermentation products - alcohols, volatile fatty acids other carbonaceous substances must be withdrawn.One way to solve this problem may be the use of microorganisms biodestructors for recycling organic impurities in the products of anaerobic biodegradation Under the proposed approach, the metabolic products (having acidic pH) of primary biotransformation of solid organic materials are used as media for the cultivation of fungi. Thus, cellulosic wastes are recycled in two successive stages. The aim of this work was to test the effectiveness of post-treatment liquid products of biodegradation of hygienic cotton wipes (common type of waste on the ISS) by the fungus Trichoderma viridae under orbital flight. The study was conducted onboard biosatellite Bion -M1, where was placed a bioreactor, designed to carry

  3. Photocatalytic and photoelectrochemical hydrogen production on strontium titanate single crystals

    SciTech Connect

    Wagner, F.T.; Somorjai, G.A.

    1980-01-01

    Sustained photogeneration of hydrogen was observed on metal-free as well as on platinized SrTiO/sub 3/ single crystals illuminated in aqueous alkaline electrolytes or in the presence of electrolyte films. Hydrogen evolution rates increased with electrolyte hydroxide concentration, most strongly at hydroxide concentrations above 5 N. Both stoichiometric and prereduced metal-free crystals were active for hydrogen photoproduction. No activity was observed from crystals in neutral or acidic solutions or in water vapor in the absence of a crust of a basic deliquescent compounds. Metal-free crystals appear to evolve hydrogen via a photocatalytic mechanism in which all chemistry occurs at the illuminated surface. The results allow direct comparison of the photocatalytic and photoelectrochemical processes and have implications for the development of heterogeneous photocatalysis at the gas-solid interface.

  4. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  5. 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.

  6. 76 FR 39897 - In the Matter of Certain Liquid Crystal Display Devices and Products Containing the Same; Notice...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-07

    ... COMMISSION In the Matter of Certain Liquid Crystal Display Devices and Products Containing the Same; Notice... the United States after importation of certain liquid crystal display devices and products containing... importation of certain liquid crystal display devices and products containing the same that infringe one...

  7. 76 FR 11512 - In the Matter of Certain Liquid Crystal Display Devices, Products Containing Same, and Methods...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-02

    ... COMMISSION In the Matter of Certain Liquid Crystal Display Devices, Products Containing Same, and Methods for... within the United States after importation of certain liquid crystal display devices, products containing... after importation of certain liquid crystal display devices, products containing same, and methods...

  8. 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.

  9. 75 FR 14470 - Enforcement Proceeding; In the Matter of Certain Liquid Crystal Display Devices and Products...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-25

    ... From the Federal Register Online via the Government Publishing Office INTERNATIONAL TRADE COMMISSION Enforcement Proceeding; In the Matter of Certain Liquid Crystal Display Devices and Products... importation, and the sale within the United States after importation of certain liquid crystal display...

  10. Identification and purification of a protein that induces production of the Lactobacillus acidophilus bacteriocin lactacin B.

    PubMed Central

    Barefoot, S F; Chen, Y R; Hughes, T A; Bodine, A B; Shearer, M Y; Hughes, M D

    1994-01-01

    Lactacin B is a heat-stable bacteriocin produced by Lactobacillus acidophilus N2 that is active against closely related lactobacilli, including Lactobacillus delbrueckii subsp. lactis (formerly Lactobacillus leichmannii) ATCC 4797. Pure producer cultures propagated in MRS broth (initial pH 6.5) contain no lactacin B; it is detected only in cultures maintained at pH 5.0 to 6.0 and produced optimally at pH 6.0 S. F. Barefoot and T. R. Klaenhammer, Antimicrob. Agents Chemother. 26:328-334, 1984). Associative growth of producer and indicator, L. delbrueckii subsp. lactis ATCC 4797, resulted in production of an inhibitor identical to lactacin B. Associative growth increased lactacin B production from nondetectable levels (< 100 activity units [AU]/ml) to between 3,200 and 6,400 AU/ml in MRS broth (initial pH 6.5) and resulted in early but equal production of lactacin B (approximately 25,600 AU/ml) in broth maintained at pH 6.0. Indicator cells, but not spent culture filtrates, induced lactacin B production. Indicator cells disrupted by a French pressure cell yielded cell-free filtrates containing inducing activity. Chromatofocusing and gel filtration high-performance liquid chromatography of cell-free filtrates yielded a protein with a pI of 4.1 and a molecular size of approximately 58 kDa that induced lactacin B production. Analytical isoelectric focusing yielded a single protein band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels contained a 28-kDa protein suggesting a two-subunit structure. Protein sequencing identified an N-terminal serine and 18 additional amino acids. To our knowledge, there are not previous descriptions of proteins that induce bacteriocin production in lactic acid bacteria. Images PMID:7986029

  11. The Production, Purification and Properties of the Biopolymer Levan Produced by the Bacterium Erwinia Herbicola

    DTIC Science & Technology

    1989-08-01

    with large-scale production in mind. In addition, films formed from the purified polymer were evaluated. Levan is an extracellular B- fructan ...Clarke, M. L., Jansson, P. E. and McNeil, K. E., Fructan from Erwlnla herblcola. J. Bact., 151(3)-.1595-1597 (1982). 23. Han, Y. W., Clarke, M. A

  12. 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.

  13. 77 FR 5055 - Certain Liquid Crystal Display Devices and Products Containing the Same; Determination Not To...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-01

    ... From the Federal Register Online via the Government Publishing Office INTERNATIONAL TRADE COMMISSION Certain Liquid Crystal Display Devices and Products Containing the Same; Determination Not To Review Initial Determination Granting Joint Motion To Terminate Based on Settlement...

  14. Recombinant production, isotope labeling and purification of ENOD40B: a plant peptide hormone.

    PubMed

    Chae, Young Kee; Tonneli, Marco; Markley, John L

    2012-08-01

    The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.

  15. Purification of triacylglycerols for biodiesel production from Nannochloropsis microalgae by membrane technology.

    PubMed

    Giorno, Filomena; Mazzei, Rosalinda; Giorno, Lidietta

    2013-07-01

    Triacylglycerols recovery from wet microalgae is a key aspect of biodiesel production, because of the energetic balance gained from avoiding biomass drying. In order to isolate TAG from Nannochloropsis cells, the possibility to concentrate biomass and to recover TAG in a single step by membrane process was studied. Different polymeric membranes were selected and screened on the basis of adsorption test and permeation flux. Results showed that membrane of regenerated cellulose (RC) with nominal molecular weight cutoff of 100 kDa and 30 kDa gave the best performance. Indeed, permeate flux was stable during ultrafiltration experiment in concentration mode and no severe fouling/cake deposition was observed. Both membranes allowed to recover permeates with high content of triacylglicerols. However, a more purity of the triacylglicerols from the other co-products was only obtained with the 30 kDa RC membrane because the retention of the unwanted proteins was in the range of 89%.

  16. Production and purification of plasmid DNA vaccines: is there scope for further innovation?

    PubMed

    Xenopoulos, Alex; Pattnaik, Priyabrata

    2014-12-01

    The demand for plasmid DNA (pDNA) has vastly increased over the past decade in response to significant advances that have been made in its application for gene therapy and vaccine development. Plasmid DNA-based vaccines are experiencing a resurgence due to success with prime-boost immunization strategies. The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host, improvements in product recovery and innovative delivery methods. This review summarizes recent advances in large-scale pDNA vaccine manufacturing, ranging from upstream processing, downstream processing and formulation, as such information is usually not available to the scientific community. The article will highlight technology gaps and offer insight on further scope of innovation.

  17. Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX)

    PubMed Central

    Campos, Bruna Medeia; Liberato, Marcelo Vizona; Polikarpov, Igor; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2015-01-01

    In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method. PMID:25760706

  18. Cloning, 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.

  19. Crystallization products of risedronate with carbohydrates and their substituted derivatives.

    PubMed

    Kos, Jiri; Pentakova, Monika; Oktabec, Zbynek; Krejcik, Lukas; Mandelova, Zuzana; Harokova, Pavla; Hruskova, Jana; Pekarek, Tomas; Dammer, Ondrej; Tkadlecova, Marcela; Havlicek, Jaroslav; Vinsova, Jarmila; Kral, Vladimir; Dohnal, Jiri; Jampílek, Josef

    2011-05-04

    The gastrointestinal absorption of bisphosphonates is in general only about 1%. To address this problem mixtures of risedronate monosodium salt with twelve varied sugar alcohols, furanoses, pyranoses and eight gluco-, manno- and galactopyranoside derivatives as counterions were designed in an effort to prepare co-crystals/new entities with improved intestinal absorption. Crystalline forms were generated by means of kinetically and/or thermodynamically controlled crystallization processes. One hundred and fifty-two prepared samples were screened by means of FT-NIR and FT-Raman spectroscopy. No co-crystal was prepared, but noteworthy results were obtained. A new solid phase of risedronate monosodium salt generated in the presence of phenyl-β-d-galactopyranoside under thermodynamically controlled crystallization conditions was found and also characterized using solid state NMR spectroscopy, X-ray powder diffraction and differential scanning calorimetry. This new polymorph was named as form P. Interactions between risedronate monosodium salt and both carbohydrates were confirmed by means of molecular dynamics simulation. In the present study the relationships between the chemical structures of the studied compounds required for crystalline form change are discussed.

  20. Improved production, purification and bioactivity of a polysaccharide from submerged cultured Ganoderma lucidum.

    PubMed

    Zhou, Huabin; Liu, Gaoqiang; Huang, Furu; Wu, Xiangting; Yang, Hailong

    2014-12-01

    Polysaccharides from Ganoderma lucidum showed multiple biological activities, such as immuno-modulating, antitumor, antioxidant, and hepatoprotective activity, etc. Adlay oil was added into the media to enhance polysaccharide production by submerged culture of G. lucidum in this work. The results revealed the optimal concentration of adlay oil was 1.5 % for polysaccharide production of G. lucidum. Analysis of the polysaccharide components confirmed that no novel components were biosynthesized by the addition of adlay oil. The main fraction of extracellular polysaccharide, GLEP-2, was isolated from the submerged culture broth of G. lucidum by ethanol precipitation, filtration, DEAE cellulose-52 and Sepharose CL-6B chromatography. GLEP-2, which was composed of glucose, galactose, mannose, arabinose, and rhamnose in a ratio of 332:55:32:13:3 respectively, had an average molecular weight of ~2.08 × 10(4) Da. The bioactivity tests demonstrated that GLEP-2 enhanced the T lymphocyte proliferation significantly at a concentration of 200 μg/mL and B lymphocyte proliferation at lower concentrations of 50 μg/mL. The results suggest polysaccharides from the submerged culture of G. lucidum are potential candidates for further development and possible commercial applications, especially in the pharmaceutical and functional foods industries.

  1. Production and purification of a solvent-resistant esterase from Bacillus licheniformis S-86.

    PubMed

    Torres, Sebastián; Baigorí, Mario D; Pandey, Ashok; Castro, Guillermo R

    2008-12-01

    New thermophilic and organic-solvent-tolerant Bacillus licheniformis S-86 strain is able to produce two active and solvent-stable esterases. Production of type I and II esterases was substantially enhanced when oils and surfactants were supplied as carbon sources. Grape oil (0.1% v/v) and Tween 20 to 60 (0.1% v/v) had enhanced enzyme production between 1.6- and 2.2-folds. Type II esterase was purified to homogeneity in a five-step procedure. This esterase was purified 76.7-fold with a specific activity of 135 U mg(-1). Molecular mass of the enzyme was estimated to be 38.4 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Type II esterase was active mostly on esters with short acyl chains, which allowed to classify the enzyme as a carboxylesterase with a K (m) of 80.2 mmol l(-1) and a V (max) of 256.4 micromol min(-1) mg(-1) for p-nitrophenyl acetate. Also, B. licheniformis S-86 type II esterase displayed activity in presence of water-miscible organic solvents at 50% concentration and stability after 1-h incubation.

  2. Functional expression and purification of recombinant Hepcidin25 production in Escherichia coli using SUMO fusion technology.

    PubMed

    Sadr, Vahideh; Saffar, Behnaz; Emamzadeh, Rahman

    2017-04-30

    Hepcidin25 is a small cysteine-rich peptide hormone known as a new class of antimicrobial peptides. The purpose of the present study was to express, purify and investigate the antibacterial properties of recombinant human hepcidin25 protein production in Escherichia coli. Human hepcidin25 gene was optimized and fused to a small ubiquitin-related modifier (SUMO) gene for higher expression. Then SUMO-hepcidin25 was cloned into the pET-32a (+) vector and expressed in E. coli Origami. The fusion protein with a molecular weight of approximately 35kDa was analyzed on SDS-PAGE gel. The highest expression was observed after 6h induction and the fusion protein consisted approximately 47% of the total cellular protein. The purified SUMO-hepcidin25 purity was determined to be higher than 95%, with a final yield of 3.9mgl(-)(1) of media. The recombinant hepcidin25 showed antibacterial activity against both Gram negative (Klebsiella pneumonia) and Gram positive (Staphylococcus aureus and Bacillus cereus) bacteria with minimum inhibitory concentrations (MICs) of 150μgml(-1), 18.7μg/ml(-1) and 37.5μg/ml(-1), respectively. These results indicated that thioredoxin and SUMO dual fusion system is an efficient production system for synthesis functional human hepcidin25.

  3. Production, Purification, and Characterization of Polygalacturonase from Mucor circinelloides ITCC 6025

    PubMed Central

    Thakur, Akhilesh; Pahwa, Roma; Singh, Smarika; Gupta, Reena

    2010-01-01

    Mucor circinelloides produced an extracellular polygalacturonase enzyme, the production of which was enhanced when various production parameters were optimized. Maximum polygalacturonase (PGase) activity was obtained in 48 h at 30°C and pH 4.0 with pectin methyl ester (1% w/v) as carbon source and a combination of casein hydrolysate (0.1% w/v) and yeast extract (0.1% w/v) as nitrogen source. The enzyme was purified to homogeneity (13.3-fold) by Sephacryl S-100 gel-filtration chromatography. Its molecular weight was 66 kDa on SDS-PAGE. The enzyme was found to have Km and Vmax values of 2.2 mM and 4.81 IU/ml at 0.1% to 0.5% (w/v) concentration of the substrate. The addition of phenolic acids (0.05 mM), metal ions such as Mn+2, Co+2, Mg+2, Fe+3, Al+3, Hg+2, and Cu+2, and thiols had inhibitory effect on the enzyme. The enzyme showed maximum activity in the presence of polygalacturonic acid (0.1% w/v) at pH 5.5 and 42°C. PMID:21048861

  4. Production, purification, and characterization of a novel galactose oxidase from Fusarium acuminatum.

    PubMed

    Alberton, Dayane; Silva de Oliveira, Luciana; Peralta, Rosane Marina; Barbosa-Tessmann, Ione Parra

    2007-06-01

    Extra-cellular production of a novel galactose oxidase from Fusarium acuminatum using submerged fermentation was studied. Glucose (1.0% w/v) was used as the sole carbon source. Maximum galactose oxidase production (approximately 4.0 U/ml) was obtained when fermentation was carried out at 25 degrees C, with orbital shaking (100 rpm) and an initial medium of pH 7.0, for 96 h, using a 2% (v/v) inoculum made from a homogenized four-day-old liquid culture, in the presence of copper, manganese, and magnesium. The enzyme was purified by one-step affinity chromatography, with a recovery of 42% of the initial activity. The purified enzyme ran as a single band of 66 kDa in SDS-PAGE. Optimal pH and temperature for the enzyme activity were 8.0 and 30 degrees C, respectively. The enzyme was thermoinactivated at temperatures above 60 degrees C. The purified enzyme was active toward various substrates, including galactose, dihydroxyacetone, guar gum, lactose, melibiose, methyl-galactopyranoside, and raffinose. SDS was an inhibitor but EDTA, Tween 80, NH(4)(+), Na(+), Mg(2+), K(+), and glycerol were not. The Michaelis-Menten constant (K(m)) for galactose was estimated to be 16.2 mM, while maximal velocity (V(max)) was 0.27 micromol of H(2)O(2) . ml(-1) . min(-1).

  5. Preparative Purification of Liriodendrin from Sargentodoxa cuneata by Macroporous Resin

    PubMed Central

    Li, Di-Hua; Wang, Yan; Lv, Yuan-Shan; Liu, Jun-Hong; Yang, Lei; Zhang, Shu-Kun; Zhuo, Yu-Zhen

    2015-01-01

    The preparative purification of liriodendrin from Sargentodoxa cuneata using macroporous resin combined with crystallization process was evaluated. The properties of adsorption/desorption of liriodendrin on eight macroporous resins were investigated systematically. X-5 resin was selected as the most suitable medium for liriodendrin purification. The adsorption of liriodendrin on X-5 resin fitted well with the pseudo-second-order kinetic model and Langmuir isotherm model. Dynamic adsorption/desorption tests were performed using a glass column packed with X-5 resin to optimize the separation process of liriodendrin. After one treatment with X-5 resin, the content of liriodendrin in the product was increased 48.73-fold, from 0.85% to 41.42%, with a recovery yield of 88.9%. 97.48% liriodendrin was obtained by further crystallization and determined by HPLC. The purified product possessed strong antioxidant activity. In conclusion, purification of liriodendrin might expend its further pharmacological researches and further applications in pharmacy. PMID:26236742

  6. Production scale purification of Ge-68 and Zn-65 from irradiated gallium metal.

    PubMed

    Fitzsimmons, Jonathan M; Mausner, Leonard

    2015-07-01

    Germanium-68 (Ge-68) is produced by proton irradiation of a gallium metal target, purified by organic extraction and used in a medical isotope generator to produce Gallium-68 PET imaging agents. The purpose of this work was to implement a production scale separation of Ge-68 and Zn-65 that does not use organic solvents and uses a limited number of columns. The current separation approach was modified to use AG1 resin and/or Sephadex(©) G25 with zinc spikes to purify Ge-68 with near quantitative recovery. The purified Ge-68 meets DOE specifications. Methods utilizing zinc spikes resulted in the purist Ge-68 produced at Brookhaven National Lab with no other impurities by ICP-OES. During process optimization approximately 2.5 Ci of Ge-68 was purified utilizing the different processing methods, and the material was sold to the Nuclear Medicine community between 2012-2013.

  7. Industrial Production of Therapeutic Proteins: Cell Lines, Cell Culture, and Purification

    NASA Astrophysics Data System (ADS)

    Zhu, Marie M.; Mollet, Michael; Hubert, Rene S.

    The biotechnology and pharmaceutical industries have seen a recent surge in the development of biological drug products manufactured from engineered mammalian cell lines. Since the hugely successful launch of human tissue plasminogen activator in 1987 and erythropoietin in 1988, the biopharmaceutical market has grown immensely. Global sales in 2003 exceeded US 30 billion.1 Currently, a total of 108 biotherapeutics are approved and available to patients (Table 32.1). In addition, 324 medically related, biotechnology-derived medicines for nearly 150 diseases are in clinical trials or under review by the U.S. Food and Drug Administration.2 These biopharmaceutical candidates promise to bring more and better treatments to patients. Compared to small molecule drugs, biotherapeutics show exquisite specificity with fewer off-target interactions and improved safety profiles.

  8. Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies.

    PubMed Central

    Webb, M A; Lindell, J S

    1993-01-01

    Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max L.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extract of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase. PMID:8290630

  9. [Biomass production and biological purification of distillation slops in a 2-step process].

    PubMed

    Waehner, R S; Giulietti, A M; Fraile, E R

    1983-01-01

    In order to lower the chemical demand (COD) of slops from cane molasses alcohol a treatment of two steps which allows the production of single cell protein of Candida utilis and Paecilomyces variotii has been performed. Its use reduces the treatment cost. In the first step the slops without sterilization supplemented with ammonium sulphate (5 g.l-1) and dipotassium phosphate (0.5 g.l-1) was inoculated with C. utilis and P. variotii. The yield was 24 and 18 g.l-1 of dry biomass and COD reduction of 36 and 75% respectively. In the second step, the remainder effluents were treated with Aspergillus niger. The final COD reduction attained was 93 and 92% respectively.

  10. 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.

  11. Production, Purification, and Identification of Cholest-4-en-3-one Produced by Cholesterol Oxidase from Rhodococcus sp. in Aqueous/Organic Biphasic System

    PubMed Central

    Wu, Ke; Li, Wei; Song, Jianrui; Li, Tao

    2015-01-01

    Cholest-4-en-3-one has positive uses against obesity, liver disease, and keratinization. It can be applied in the synthesis of steroid drugs as well. Most related studies are focused on preparation of cholest-4-en-3-one by using whole cells as catalysts, but production of high-quality cholest-4-en-3-one directly from cholesterol oxidase (COD) using an aqueous/organic two-phase system has been rarely explored. This study set up an enzymatic reaction system to produce cholest-4-en-3-one. We developed and optimized the enzymatic reaction system using COD from COX5-6 (a strain of Rhodococcus) instead of whole-cell biocatalyst. This not only simplifies and accelerates the production but also benefits the subsequent separation and purification process. Through extraction, washing, evaporation, column chromatography, and recrystallization, we got cholest-4-en-3-one with purity of 99.78%, which was identified by nuclear magnetic resonance, mass spectroscopy, and infrared spectroscopy. In addition, this optimized process of cholest-4-en-3-one production and purification can be easily scaled up for industrial production, which can largely decrease the cost and guarantee the purity of the product. PMID:25733914

  12. Semiconductor Grade, Solar Silicon Purification Project. [photovoltaic solar energy conversion

    NASA Technical Reports Server (NTRS)

    Ingle, W. M.; Rosler, R. S.; Thompson, S. W.; Chaney, R. E.

    1979-01-01

    A low cost by-product, SiF4, is reacted with mg silicon to form SiF2 gas which is polymerized. The (SiF2)x polymer is heated forming volatile SixFy homologues which disproportionate on a silicon particle bed forming silicon and SiF4. The silicon analysis procedure relied heavily on mass spectroscopic and emission spectroscopic analysis. These analyses demonstrated that major purification had occured and some samples were indistinguishable from semiconductor grade silicon (except possibly for phosphorus). However, electrical analysis via crystal growth reveal that the product contains compensated phosphorus and boron.

  13. HD gas purification for polarized HDice targets production at Jefferson Lab

    SciTech Connect

    Whisnant, Charles; D'Angelo, Annalisa; Colaneri, Luca; Devilbiss, J; Kageya, Tsuneo; Loving, D A; Lowry, Michael; Rizzo, Alessandro; Sandorfi, Andrew; Schaerf, Carlo; Storey, J D; Wallace, C M; Wei, Xiangdong; Zonta, Irene

    2014-06-01

    Solid, frozen-spin targets of molecular HD were rst developed for nuclear physics by a collaboration between Syracuse University and Brookhaven National Lab. They have been successfully used in measurements with photon beams, rst at the Laser-Electron-Gamma-Source [1] and most recently at Je erson Lab during the running of the E06-101 (g14) experiment [2]. Preparations are underway to utilize the targets in future electron experiments after the completion of the 12 GeV JLab upgrade [3]. HD is an attractive target since all of the material is polarizable, of low Z, and requires only modest holding elds. At the same time, the small contributions from the target cell can be subtracted from direct measurements. Reaching the frozen-spin state with both high polarization and a signi cant spin relaxation time requires careful control of H2 and D2 impurities. Commercially available HD contains 0.5 - 2% concentrations of H2 and D2. Low-temperature distillation is required to reduce these concentrations to the 104 level to enable useful target production. This distillation is done using a column lled with heli-pack C [4] to give good separation e ciency. Approximately 12 moles of commercial HD is condensed into the mechanically refrigerated system at the base temperature of 11K. The system is then isolated and the temperature stabilized at 18K producing liquid HD, which is boiled by a resistive heater. The circulation established by the boil-o condensing throughout the column then ltering back down produces a steady-state isotopic separation permitting the extraction of HD gas with very low H2 and D2 content. A residual gas analyzer initially monitors distillation. Once the H2 concentration falls below its useful operating range, samples are periodically collected for analysis using gas chromatography [5] and Raman scattering. Where the measurement techniques overlap, good agreement is obtained. The operation of the distillery and results of gas analysis will be discussed

  14. Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences.

    PubMed

    Carvalho, Rimenys Junior; Cabrera-Crespo, Joaquin; Tanizaki, Martha Massako; Gonçalves, Viviane Maimoni

    2012-05-01

    Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.

  15. The LUCIFER project and production issues for crystals needed in rare events physics experiments

    NASA Astrophysics Data System (ADS)

    Dafinei, I.

    2014-05-01

    The detection of elusive particles and in general the construction of detectors with high sensitivity for applications in the physics of rare events requires the use of new high quality crystals with enhanced characteristics. The production of such materials often depends upon the application of dedicated methods for the entire production process from synthesis of raw materials up to the storage and transport of the finished product ready for use for the construction of the particle detector. Cryogenic bolometers and the more sophisticated scintillating bolometers are among the most promising detectors used in rare event physics, particularly in Neutrinoless Double Beta Decay (0νDBD) experiments. Operated at extremely low temperatures (≈10 mK) such devices need high purity crystals with a very high crystal perfection and low level of intrinsic radioactivity. Moreover, in the case of 0νDBD application, the crystal requires the presence of the nuclide of interest in a sufficient amount i.e. isotope enriched materials are employed. The current work reviews scientific and technological aspects related to the crystal production for rare events physics experiments, particularly for bolometric application. In the case of enriched isotopes used in 0νDBD experiments, the problems related to a maximum production yield are stressed. The discussion is illustrated with results obtained in the activities connected to the procurement of ZnSe crystals for the experiment Low-background Underground Cryogenic Installation For Elusive Rates (LUCIFER).

  16. 75 FR 6705 - In the Matter of: Certain Liquid Crystal Display Modules and Products Containing the Same, and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-10

    ... COMMISSION In the Matter of: Certain Liquid Crystal Display Modules and Products Containing the Same, and... importation, and the sale within the United States after importation of certain liquid crystal display modules... importation of liquid crystal display modules or products containing the same, or methods for making the...

  17. 75 FR 445 - In the Matter of Certain Liquid Crystal Display Devices and Products Containing the Same; Notice...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-05

    ... Crystal Display Devices and Products Containing the Same; Notice of Investigation AGENCY: U.S... importation, and the sale within the United States after importation of certain liquid crystal display devices... crystal display devices or products containing same that infringe one or more of claims 1, 3, and 6 of...

  18. Production, purification and characterization of an extracellular alpha-amylase enzyme isolated from Aspergillus flavus.

    PubMed

    Abou-Zeid, A M

    1997-01-01

    Filamentous fungi isolated from cereals were screened for their ability to produce alpha-amylase (1,4-alpha-glucan glucanohydrolase, EC 3.2.1.1). A selected strain identified as Aspergillus flavus showed high enzymatic activity. A single extracellular alpha-amylase was purified to homogeneity by a starch adsorption method. The molecular weight (M(r)) of the A. flavus alpha-amylase was approximately 75,000 +/- 3,000 by polyacrylamide gel electrophoresis (PAGE) and that of the subunit was approximately 75,000 +/- 3000 SDS-PAGE. The optimal activity of the purified enzyme was achieved at pH 7.0 and 30 degrees C. K+ ions increased the alpha-amylase activity, but Mg2+ did not greatly affect enzyme activity. Mn2+, Zn2+, Cu2+ and Fe3+ ions strongly inhibited the enzyme activity. The products of hydrolysis of native starch by the A. flavus enzyme were mainly glucose as well as unidentified oligosaccharides.

  19. Lipolytic Potential of Aspergillus japonicus LAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

    PubMed Central

    Souza, Lívia Tereza Andrade; Oliveira, Jamil S.; dos Santos, Vera L.; Regis, Wiliam C. B.; Santoro, Marcelo M.; Resende, Rodrigo R.

    2014-01-01

    Lipolytic potential of Aspergillus japonicus LAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu2+, Ni2+, and Al3+ showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. The KM and Vmax values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications. PMID:25530954

  20. Production and purification of novel thermostable alkaline protease from Anoxybacillus sp. KP1.

    PubMed

    Matpan Bekler, F; Acer, Ö; Güven, K

    2015-09-26

    In this study, an extracellular novel alkaline protease (EC 3.4.21-24, 99) from a thermophilic and aerobic strain of Anoxybacillus sp. KP1 has been studied. Maximum protease activity was obtained at 50 degC at pH 9.0 after 24 hours of incubation. Among the carbon and nitrogen sources used; the optimum protease production was with soluble starch, maltose, urea and casamino acid. The enzyme was purified by ammonium sulphate precipitation and Sephadex G-75 gel chromatography. Molecular weight of purified enzyme was determined as 106 kDa by SDS-PAGE. Purified protease was stable at 50-60 °C and at pH 9.0 for 1 h. The enzyme activity was increased in the presence of Ca2+, Cu2+, Tween 80 and Triton X-100, however the enzyme activity was inhibited in the presence of Hg2+, ethylene diamine tetra acetic acid (EDTA) and H2O2. Proteolytic activity was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF). The enzyme seems to be a serine alkaline protease. In the presence of detergents, the protease was clearly stable and residual activity was between 73-82%.

  1. Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras.

    PubMed

    Neddersen, Mara; Elleuche, Skander

    2015-12-01

    Degradation of complex plant biomass demands a fine-regulated portfolio of glycoside hydrolases. The LE (LguI/Eco81I)-cloning approach was used to produce two enzyme chimeras CB and BC composed of an endoglucanase Cel5A (C) from the extreme thermophilic bacterium Fervidobacterium gondwanense and an archaeal β-glucosidase Bgl1 (B) derived from a hydrothermal spring metagenome. Recombinant chimeras and parental enzymes were produced in Escherichia coli and purified using a two-step affinity chromatography approach. Enzymatic properties revealed that both chimeras closely resemble the parental enzymes and physical mixtures, but Cel5A displayed lower temperature tolerance at 100°C when fused to Bgl1 independent of the conformational order. Moreover, the determination of enzymatic performances resulted in the detection of additive effects in case of BC fusion chimera. Kinetic measurements in combination with HPLC-mediated product analyses and site-directed mutation constructs indicated that Cel5A was strongly impaired when fused at the N-terminus, while activity was reduced to a slighter extend as C-terminal fusion partner. In contrast to these results, catalytic activity of Bgl1 at the N-terminus was improved 1.2-fold, effectively counteracting the slightly reduced activity of Cel5A by converting cellobiose into glucose. In addition, cellobiose exhibited inhibitory effects on Cel5A, resulting in a higher yield of cellobiose and glucose by application of an enzyme mixture (53.1%) compared to cellobiose produced from endoglucanase alone (10.9%). However, the overall release of cellobiose and glucose was even increased by catalytic action of BC (59.2%). These results indicate possible advantages of easily produced bifunctional fusion enzymes for the improved conversion of complex polysaccharide plant materials.

  2. Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies.

    PubMed

    Hu, Zhonglan; Chen, Zhenyu; Huang, Nongyu; Teng, Xiu; Zhang, Jun; Wang, Zhen; Wei, Xiaoqiong; Qin, Ke; Liu, Xiao; Wu, Xueping; Tang, Huan; Zhu, Xiaofeng; Cui, Kaijun; Li, Jiong

    2015-03-01

    Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl β-D-1-thio galactoside (IPTG) at 37°C for 4h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined.

  3. New Lipase for Biodiesel Production: Partial Purification and Characterization of LipSB 25-4

    PubMed Central

    Ayaz, Berk; Ceylan, Ozgur; Okmen, Gulten

    2014-01-01

    The lipolytic activities of 300 Streptomyces isolates were determined in Tributyrin and Rhodamine-B Agar. Lipase activities were also measured with p-nitrophenyl palmitate (p-NPP) as a substrate. The strain of Streptomyces bambergiensis OC 25-4 used in this study was selected among 300 strains of Streptomyces from MUCC as the best lipase producer. The incubation conditions were optimized and the inoculum amount, incubation period, effect of carbon and nitrogen sources, and rates of MgSO4 and CaCO3 were investigated. LipSB 25-4 (the lipase produced by S. bambergiensis OC 25-4 strain) was partially purified with ammonium sulphate precipitation, dialysis, and gel filtration chromatography 2.73-fold and with 92.12 U/mg specific activity. The optimal pH and temperature for LipSB 25-4 were determined as 8.0 and 50°C, respectively. The lipase has high stability in all pH and temperature values used in this study. While LipSB 25-4 was slightly activated in the presence of β-mercaptoethanol, it was slightly reduced by PMSF. The enzyme conserved approximately 75% of its activity at the end of 60 h, in the presence of methanol and ethanol. Since LipSB 25-4 displays high activity in the thermophilic conditions and stability in the presence of organic solvents, this lipase can catalyse the biodiesel production from olive oil by the transesterification reactions. PMID:25937966

  4. Single step purification of concanavalin A (Con A) and bio-sugar production from jack bean using glucosylated magnetic nano matrix.

    PubMed

    Kim, Ho Myeong; Cho, Eun Jin; Bae, Hyeun-Jong

    2016-08-01

    Jack bean (JB, Canavalia ensiformis) is the source of bio-based products, such as proteins and bio-sugars that contribute to modern molecular biology and biomedical research. In this study, the use of jack bean was evaluated as a source for concanavalin A (Con A) and bio-sugar production. A novel method for purifying Con A from JBs was successfully developed using a glucosylated magnetic nano matrix (GMNM) as a physical support, which facilitated easy separation and purification of Con A. In addition, the enzymatic conversion rate of 2% (w/v) Con A extracted residue to bio-sugar was 98.4%. Therefore, this new approach for the production of Con A and bio-sugar is potentially useful for obtaining bio-based products from jack bean.

  5. 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

  6. Efficient biosynthesis of D-allose from D-psicose by cross-linked recombinant L-rhamnose isomerase: separation of product by ethanol crystallization.

    PubMed

    Menavuvu, Buetusiwa Thomas; Poonperm, Wayoon; Leang, Khim; Noguchi, Naoki; Okada, Hiromi; Morimoto, Kenji; Granström, Tom Birger; Takada, Goro; Izumori, Ken

    2006-04-01

    Mass production of a rare aldohexose D-allose from D-psicose was achieved in a batch reaction by crude recombinant L-rhamnose isomerase (L-RhI) cross-linked with glutaraldehyde. The D-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose D-fructose by immobilized recombinant D-tagatose 3-epimerase (D-TE). At an equilibrium state, 25% of D-psicose was isomerized to D-allose, that is, 25 g of D-allose was obtained from 100 g of D-psicose. The D-allose product was easily separated and crystallized from the reaction mixture that contains 25%D-allose, 8%D-altrose and 67%D-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure D-allose crystals was produced from 1.5 kg of D-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages.

  7. High productivity purification of immunoglobulin G monoclonal antibodies on starch-coated magnetic nanoparticles by steric exclusion of polyethylene glycol.

    PubMed

    Gagnon, Pete; Toh, Phyllicia; Lee, Jeremy

    2014-01-10

    We achieved exceptionally high capacity capture of monoclonal IgG by adding 200 nm starch-coated magnetic particles as nucleation centers, adding polyethylene glycol (PEG), then collecting the particle-associated antibody in a magnetic field. Experimental data suggest that accretion of IgG begins on particle surfaces then continues with fusion of particle-centric accretions up to about 1mm in a process that closely parallels PEG precipitation. An embedded nanoparticle mass of 1.3% of the IgG mass is adequate to enable efficient magnetic collection of the associated IgG. Recovery of purified IgG averaged 98% up to loads of 78 mg of IgG per mg of particles. Converted to an equivalent volume of settled particles, this represents about 58 g IgG per mL of nanoparticles, which is roughly 1000 times higher than the average capacity of commercial protein A porous particles packed in columns. When applied to cell culture harvest clarified by centrifugation and microfiltration, performing the nanoparticle technique under physiological conditions permitted only a 10-fold reduction of host cell protein (HCP) contamination and IgG recovery less than 50%. Application of a more capable clarification method and operating the nanoparticle method at 0.5-1.0M NaCl supported more than 99% HCP reduction and 87% IgG recovery. The high salt concentration also dramatically diminished the influence of operating pH on selectivity. The nanoparticle step was followed by sample application without buffer exchange to a column packed with multimodal electropositive-hydrophobic particles that reduced HCP to 2 ppm. Aggregate content was reduced from 4.9 to 3.6% at the nanoparticle step, then to less than 0.05% at the multimodal step. The multimodal step also removed residual PEG. Overall IgG recovery was 69%. The ability of the system to achieve purity similar to protein A, but dramatically higher productivity than packed columns, suggests that the technique could evolve as a credible option for

  8. Production, Purification, and Properties of a Thermostable beta-Glucosidase from a Color Variant Strain of Aureobasidium pullulans.

    PubMed

    Saha, B C; Freer, S N; Bothast, R J

    1994-10-01

    A color variant strain of Aureobasidium pullulans (NRRL Y-12974) produced beta-glucosidase activity when grown in liquid culture on a variety of carbon sources, such as cellobiose, xylose, arabinose, lactose, sucrose, maltose, glucose, xylitol, xylan, cellulose, starch, and pullulan. An extracellular beta-glucosidase was purified 129-fold to homogeneity from the cell-free culture broth of the organism grown on corn bran. The purification protocol included ammonium sulfate treatment, CM Bio-Gel A agarose column chromatography, and gel filtrations on Bio-Gel A-0.5m and Sephacryl S-200. The beta-glucosidase was a glycoprotein with native molecular weight of 340,000 and was composed of two subunits with molecular weights of about 165,000. The enzyme displayed optimal activity at 75 degrees C and pH 4.5 and had a specific activity of 315 mumol . min . mg of protein under these conditions. The purified beta-glucosidase was active against p-nitrophenyl-beta-d-glucoside, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose, with K(m) values of 1.17, 1.00, 0.34, 0.36, 0.64, 0.68, and 1.65 mM, respectively. The enzyme activity was competitively inhibited by glucose (K(i) = 5.65 mM), while fructose, arabinose, galactose, mannose, and xylose (each at 56 mM) and sucrose and lactose (each at 29 mM) were not inhibitory. The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol (7.5%, vol/vol) stimulated the initial enzyme activity by 15%. Glucose production was enhanced by 7.9% when microcrystalline cellulose (2%, wt/vol) was treated for 48 h with a commercial cellulase preparation (5 U/ml) that was supplemented with the purified beta-glucosidase (0.21 U/ml) from A. pullulans.

  9. Expression, purification, crystallization and preliminary X-ray crystallographic studies of the trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45.

    PubMed

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

    2005-01-01

    The trehalulose synthase (MutB) from Pseudomonas mesoacidophila MX-45, belonging to glycoside hydrolase family 13, catalyses the isomerization of sucrose to trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (alpha-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 A resolution using synchrotron radiation and belongs to space group P1, with unit-cell parameters a = 63.8, b = 72.0, c = 82.2 A, alpha = 67.5, beta = 73.1, gamma = 70.8 degrees, while the other form diffracts to 1.8 A resolution using synchrotron radiation and belongs to space group P2(1), with unit-cell parameters a = 63.7, b = 85.9, c = 119.7 A, beta = 97.7 degrees. A molecular-replacement solution has been found using the structure of the isomaltulose synthase (PalI) from Klebsiella sp. LX3 as a search model.

  10. Expression, purification, crystallization and preliminary X-ray crystallographic studies of the trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45

    PubMed Central

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

    2005-01-01

    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 P21, 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. PMID:16508103

  11. 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.

  12. Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content.

    PubMed

    Figueiredo, Douglas B; Carvalho, Eneas; Santos, Mauricio P; Kraschowetz, Stefanie; Zanardo, Rafaela T; Campani, Gilson; Silva, Gabriel G; Sargo, Cíntia R; Horta, Antonio Carlos L; de C Giordano, Roberto; Miyaji, Eliane N; Zangirolami, Teresa C; Cabrera-Crespo, Joaquin; Gonçalves, Viviane Maimoni

    2017-03-01

    Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.

  13. Ferroelectric liquid crystal SLMs: from prototypes to products

    NASA Astrophysics Data System (ADS)

    O'Callaghan, Michael J.; Handschy, Mark A.

    2001-11-01

    The road from a new technology's proof-of-principle prototype to commercially successful products always seems to be more challenging, more expensive, and longer than its inventors had imagined. Displaytech built its first experimental FLC-VLSI SLMs in 1989, began ramping up its efforts to commercialize FLC-VLSI displays around 1995, and now is building more than 100,000 displays per month with its manufacturing partner Miyota. Here we review the motivation for using FLC-VLSI technology and trace the developments that were necessary for its commercialization. We discuss problems that had to be overcome in FLC materials, device design, manufacturing, applications, product definition, and systems support in order to develop the technology and to lower barriers to its adoption by customers. The principal focus here is on technical challenges encountered in manufacturing and in FLC materials development that had to be met to go from hand-built prototypes to mass production. We also review future potential markets other than displays and describe some of our work on experimental FLC-VLSI devices that addresses those opportunities. Examples include holographic optical data storage, 3D projection, optical image processing, smart-pixel SLMs, and high-speed computer interfaces needed to support high frame rate SLMs.

  14. Extraction and purification of high added value compounds from by-products of the winemaking chain using alternative/non-conventional processes/technologies.

    PubMed

    Yammine, Sami; Brianceau, Sylène; Manteau, Sébastien; Turk, Mohammad; Ghidossi, Rémy; Vorobiev, Eugène; Mietton-Peuchot, Martine

    2016-12-21

    Grape byproducts are today considered as a cheap source of valuable compounds since existent technologies allow the recovery of target compounds and their recycling. The goal of the current article is to explore the different recovery stages used by both conventional and alternative techniques and processes. Alternative pre-treatments techniques reviewed are: ultrasounds, pulsed electric fields and high voltage discharges. Additionally, non-conventional solvent extraction under high pressure, specifically, supercritical fluid extraction and subcritical water extraction are discussed. Finally alternative purification technologies, for example membrane processing were also examined. The intent is to describe the mechanisms involved by these alternative technologies and to summarize the work done on the improvement of the extraction process of phenolic compounds from winery by-products. With a focus on the developmental stage of each technology, highlighting the research need and challenges to be overcome for an industrial implementation of these unitary operations in the overall extraction process. A critical comparison of conventional and alternative techniques will be reviewed for ethe pre-treatment of raw material, the diffusion of polyphenols and the purification of these high added value compounds. This review intends to give the reader some key answers (costs, advantages, drawbacks) to help in the choice of alternative technologies for extraction purposes.

  15. Fermentative production of poly (γ-glutamic acid) from renewable carbon source and downstream purification through a continuous membrane-integrated hybrid process.

    PubMed

    Kumar, Ramesh; Pal, Parimal

    2015-02-01

    Experimental investigations were carried out on continuous and direct production of poly-(γ-glutamic acid) in a hybrid reactor system that integrated conventional fermentative production step with membrane-based downstream separation and purification. Novelty of the integrated system lies in high degree of purity, conversion, yield and productivity of poly-(γ-glutamic acid) through elimination of substrate-product inhibitions of traditional batch production system. This new system is compact, flexible, eco-friendly and largely fouling-free ensuring steady and continuous production of poly-(γ-glutamic acid) directly from a renewable carbon source at the rate of 0.91 g/L/h. Cross-flow microfiltration membrane modules ensured almost complete separation and recycle of cells without much fouling problem. Well-screened ultrafiltration membrane module helped to concentrate poly-(γ-glutamic acid) while ensuring recovery and recycle of 96% unconverted carbon source resulting in yield of 0.6g/g along with high product purity.

  16. 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.

  17. 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.

  18. Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu

    SciTech Connect

    Kondou, Youhei; Kitazawa, Daisuke; Takeda, Shigeki; Yamashita, Eiki; Mizuguchi, Mineyuki; Kawano, Keiichi; Tsukihara, Tomitake

    2005-01-01

    Bacteriophage Mu baseplate protein gene product 44 was crystallized. The crystal belongs to space group R3, with unit-cell parameters a = b = 126.6, c = 64.2 Å. Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-rays to at least 2.1 Å resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.

  19. 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.

  20. 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°.

  1. 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}.

  2. 76 FR 30968 - In the Matter of Certain Liquid Crystal Display Devices, Products Containing Same, and Methods...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-27

    ... From the Federal Register Online via the Government Publishing Office INTERNATIONAL TRADE COMMISSION In the Matter of Certain Liquid Crystal Display Devices, Products Containing Same, and Methods for... importation of certain liquid crystal display (``LCD'') devices, products containing same, and methods...

  3. 75 FR 14470 - In the Matter of: Certain Liquid Crystal Display Modules and Products Containing the Same, and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-25

    ... From the Federal Register Online via the Government Publishing Office INTERNATIONAL TRADE COMMISSION In the Matter of: Certain Liquid Crystal Display Modules and Products Containing the Same, and... importation of certain liquid crystal display modules, products containing the same, and methods for...

  4. 75 FR 13780 - In the Matter of Certain Liquid Crystal Display Devices and Products Containing the Same; Notice...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-23

    ... From the Federal Register Online via the Government Publishing Office INTERNATIONAL TRADE COMMISSION In the Matter of Certain Liquid Crystal Display Devices and Products Containing the Same; Notice... crystal display modules, products containing the same, and methods for making the same by reason...

  5. Purification, crystallization and preliminary X-ray analysis of rice BGlu1 β-glucosidase with and without 2-deoxy-2-fluoro-β-d-glucoside

    SciTech Connect

    Chuenchor, Watchalee; Pengthaisong, Salila; Yuvaniyama, Jirundon; Opassiri, Rodjana; Svasti, Jisnuson; Ketudat Cairns, James R.

    2006-08-01

    Rice BGlu1 β-glucosidase was purified from recombinant E. coli and crystallized with and without the inhibitor 2-deoxy-2-fluoro-β-d-glucose. The crystals diffracted to 2.15 and 2.75 Å, respectively. Rice (Oryza sativa) BGlu1 β-glucosidase was expressed in Escherichia coli with N-terminal thioredoxin and hexahistidine tags and purified by immobilized metal-affinity chromatography (IMAC). After removal of the N-terminal tags, cation-exchange and S-200 gel-filtration chromatography yielded a 50 kDa BGlu1 with >95% purity. The free enzyme and a complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-d-glucopyranoside inhibitor were crystallized by microbatch and hanging-drop vapour diffusion. Small tetragonal crystals of BGlu1 with and without inhibitor grew in 18%(w/v) PEG 8000 with 0.1 M sodium cacodylate pH 6.5 and 0.2 M zinc acetate. Crystals of BGlu1 with inhibitor were streak-seeded into 23%(w/v) PEG MME 5000, 0.2 M ammonium sulfate, 0.1 M MES pH 6.7 to yield larger crystals. Crystals with and without inhibitor diffracted to 2.15 and 2.75 Å resolution, respectively, and had isomorphous orthorhombic unit cells belonging to space group P2{sub 1}2{sub 1}2{sub 1}.

  6. High-level production, solubilization and purification of synthetic human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1.

    PubMed

    Ren, Hui; Yu, Daoyong; Ge, Baosheng; Cook, Brian; Xu, Zhinan; Zhang, Shuguang

    2009-01-01

    Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two

  7. SnRK2.6/OST1 from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis of K50N and D160A mutants

    PubMed Central

    Yunta, Cristina; Martinez-Ripoll, Martin; Albert, Armando

    2011-01-01

    The SnRK2.6 (SNF1-related kinase 2.6) gene from Arabidopsis thaliana encodes the serine/threonine protein kinase SnRK2.6/OST1 (OPEN STOMATA 1). It plays a central role in the drought-tolerance mechanism. OST1 is in fact the main positive effector in the hydric stress response. The SnRK2.6 gene was cloned into the pGEX4T1 plasmid, mutated and expressed in Escherichia coli, allowing purification to homogeneity in two chromatographic steps. Various OST1 mutants yielded crystals using vapour-diffusion techniques, but only one mutant showed a good diffraction pattern. Its crystals diffracted to 2.8 Å resolution and belonged to space group P2221, with unit-cell parameters a = 77.7, b = 99.4, c = 108.4 Å. A promising molecular-replacement solution was found using the structure of the kinase domain of the yeast AMP-activated protein kinase SNF1 (PDB entry 3hyh) as the search model. PMID:21393844

  8. Cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacterium Bartonella henselae strain Houston-1 at 2.1 Å resolution

    PubMed Central

    Naqvi, Kubra F.; Staker, Bart L.; Dobson, Renwick C. J.; Serbzhinskiy, Dmitry; Sankaran, Banumathi; Myler, Peter J.; Hudson, André O.

    2016-01-01

    The enzyme dihydrodipicolinate synthase catalyzes the committed step in the synthesis of diaminopimelate and lysine to facilitate peptidoglycan and protein synthesis. Dihydrodipicolinate synthase catalyzes the condensation of l-aspartate 4-semialdehyde and pyruvate to synthesize l-2,3-dihydrodipico­linate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are presented. Protein crystals were grown in conditions consisting of 20%(w/v) PEG 4000, 100 mM sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.10 Å resolution. They belonged to space group P212121, with unit-cell parameters a = 79.96, b = 106.33, c = 136.25 Å. The final R values were R r.i.m. = 0.098, R work = 0.183, R free = 0.233. PMID:26750477

  9. Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications.

    PubMed

    Jugder, Bat-Erdene; Lebhar, Helene; Aguey-Zinsou, Kondo-Francois; Marquis, Christopher P

    2016-01-01

    The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.

  10. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the collagen-binding region of RspB from Erysipelothrix rhusiopathiae

    PubMed Central

    Devi, Aribam Swarmistha; Ogawa, Yohsuke; Shimoji, Yoshihiro; Ponnuraj, Karthe

    2010-01-01

    RspB is a surface adhesin of Erysipelothrix rhusiopathiae. A recombinant form of the collagen-binding region of this protein, RspB(31–348), has been overexpressed in Escherichia coli in native and selenomethionine-derivative forms and purified using affinity and gel-permeation chromatography. Thin plate-like crystals were obtained by the hanging-drop vapour-diffusion method using the same condition for both forms. The native crystals diffracted to a resolution of 2.5 Å using an in-house X-ray source, while the selenomethionine-derivative crystals diffracted to a resolution of 2.2 Å using synchrotron radiation. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 46.19, b = 66.65, c = 101.72 Å, β = 94.11°. PMID:20124711

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  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. 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.

  19. Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle).

    PubMed

    Torres-Acosta, Mario A; Aguilar-Yáñez, José M; Rito-Palomares, Marco; Titchener-Hooker, Nigel J

    2016-01-01

    Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two-phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques.

  20. Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two‐phase extraction (with and without PEG recycle)

    PubMed Central

    Torres‐Acosta, Mario A.; Aguilar‐Yáñez, José M.; Rito‐Palomares, Marco

    2015-01-01

    Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two‐phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:126–133, 2016 PMID:26561271

  1. 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.

  2. 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.

  3. Purification, crystallization and preliminary X-ray characterization of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

    PubMed

    Albillos, Silvia M; Jin, Tengchuan; Howard, Andrew; Zhang, Yuzhu; Kothary, Mahendra H; Fu, Tong-Jen

    2008-07-09

    The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond ( Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 A and belong to the tetragonal space group P4(1)22, with unit cell parameters of a = b = 150.912 A, c = 165.248 A. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

  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 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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%.

  10. Overproduction, purification, crystallization and preliminary X-ray characterization of a novel carbohydrate-binding module of endoglucanase Cel5A from Eubacterium cellulosolvens.

    PubMed

    Luís, Ana S; Alves, Victor D; Romão, Maria J; Prates, José A M; Fontes, Carlos M G A; Najmudin, Shabir

    2011-04-01

    The anaerobic cellulolytic rumen bacterium Eubacterium cellulosolvens produces a large array of cellulases and hemicellulases that are responsible for the hydrolysis of plant cell-wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises two tandemly repeated novel carbohydrate-binding modules (CBMs) and two catalytic domains belonging to glycoside hydrolase family 5 joined by flexible linker sequences. The novel CBM located at the N-terminus of the endoglucanase has been crystallized. The crystals belonged to the hexagonal space group P6(1)22 and contained a single molecule in the asymmetric unit. The structure of the L-selenomethionine derivative has been solved by a MAD experiment on crystals that diffracted to 1.75 Å resolution.

  11. 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%.

  12. 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.

  13. 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.

  14. Overproduction, purification, crystallization and preliminary X-ray analysis of the peroxiredoxin domain of a larger natural hybrid protein from Thermotoga maritima

    SciTech Connect

    Barbey, Carole; Rouhier, Nicolas; Haouz, Ahmed; Navaza, Alda; Jacquot, Jean-Pierre

    2008-01-01

    Crystals of the peroxiredoxin domain of a larger natural hybrid protein from T. maritima were obtained which diffracted to 2.9 Å resolution on a synchrotron source. Thermotoga maritima contains a natural hybrid protein constituted of two moieties: a peroxiredoxin domain at the N-terminus and a nitroreductase domain at the C-terminus. The peroxiredoxin (Prx) domain has been overproduced and purified from Escherichia coli cells. The recombinant Prx domain, which is homologous to bacterial Prx BCP and plant Prx Q, folds properly into a stable protein that possesses biological activity. The recombinant protein was crystallized and synchrotron data were collected to 2.9 Å resolution. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 176.67, c = 141.20 Å.

  15. 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%.

  16. 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.

  17. Optimization of medium components for increased production of C-phycocyanin from Phormidium ceylanicum and its purification by single step process.

    PubMed

    Singh, Niraj Kumar; Parmar, Asha; Madamwar, Datta

    2009-02-01

    Phycocyanin is a major protein produced by cyanobacteria, but very few phycocyanin-producing strains have been reported. In the present study, response surface methodology (RSM) involving a central composite design for four factors was successfully employed to optimize medium components for increased production of phycocyanin from Phormidium ceylanicum. The production of phycocyanin and interactions between sodium nitrate, calcium chloride, trace metal mix and citric acid stock were investigated and modeled. Under optimized condition P. ceylanicum was able to give 2.3-fold increase in phycocyanin production in comparison to commonly used BG 11 medium in 32 days. We have demonstrated the extraction, purification and characterization of C-phycocyanin using novel method based on filtration and single step chromatography. The protein was extracted by repeated freeze-thaw cycles and the crude extract was filtered and concentrated in stirred ultrafiltration cell (UFC). The UFC concentrate was then subjected to a single ion exchange chromatographic step. A purity ratio of 4.15 was achieved from a starting value of 1.05. The recovery efficiency of C-phycocyanin from crude extract was 63.50%. The purity was checked by electrophoresis and UV-Vis spectroscopy.

  18. Purification, crystallization and preliminary X-ray analysis of the soluble domain of the Na+-pumping cytochrome bo quinol oxidase from Vitreoscilla.

    PubMed

    Kim, Kyung Jin; Kim, Youngchang; Park, Kyung Won; Webster, Dale A; Howard, Andrew J

    2002-08-01

    The 24 kDa CyoA soluble domain of Vitreoscilla cytochrome bo quinol oxidase, which pumps out Na(+) during respiration, has been crystallized from a solution of 2 M ammonium sulfate and 5% 2-propanol. The crystal belongs to cubic space group P4(3)32, with unit-cell parameters a = b = c = 122.2 A, alpha = beta = gamma = 90 degrees and one subunit in the asymmetric unit. A 99.8% complete data set to 3.3 A has been collected at the 17-ID beamline of the Advanced Photon Source. The structure was determined by molecular replacement and refinement is in progress.

  19. 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}.

  20. PREPARATION AND PURIFICATION OF SILICON CARBIDE.

    DTIC Science & Technology

    the materials were divided into two parts. Part I covers problems of silicon carbide preparation and the growing of silicon carbide single crystals...and thin films for semiconductor devices. Part II treats problems of purity, including the purification and chemical analysis of silicon carbide and of starting materials for silicon carbide preparation.