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Sample records for cloning purification crystallization

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

    Energy Technology Data Exchange (ETDEWEB)

    Felts, Richard L. [Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Reilly, Thomas J. [Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211 (United States); Veterinary Medical Diagnostic Laboratory, University of Missouri-Columbia, Columbia, MO 65211 (United States); Calcutt, Michael J. [Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211 (United States); Tanner, John J., E-mail: tannerjj@missouri.edu [Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States)

    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.

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

    Science.gov (United States)

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

    2006-01-01

    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 P212121, 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. PMID:16820700

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

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    Meining, Winfried, E-mail: wim@csb.ki.se [Karolinska Institutet, Department of Biosciences, Center of Structural Biochemistry (Sweden); Scheuring, Johannes; Fischer, Markus; Weinkauf, Sevil [Technische Universität München, Chemistry Department (Germany); Karolinska Institutet, Department of Biosciences, Center of Structural Biochemistry (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Vibha [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Gupta, Rakesh K. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Ram Lal Anand College, University of Delhi, Benito Juarez Road, New Delhi 110021 (India); Khare, Garima [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Salunke, Dinakar M. [National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067 (India); Tyagi, Anil K., E-mail: aniltyagi@south.du.ac.in [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India)

    2008-05-01

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

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

    Science.gov (United States)

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

    2013-11-01

    Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni(2+)-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80 Å on the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.3, b = 86.1, c = 118.2 Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.52 Å(3) Da(-1) and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement.

  6. Cloning, Expression, Purification and Crystallization of NHR3 Domain from Acute Myelogenous Leukemia-related Protein AML1-ETO

    Institute of Scientific and Technical Information of China (English)

    Hai-Tao YANG; Dong-Hui WU; Xiao-Yu XUE; Wen-Xue LIANG; Xiao-Yu MIAO; Hai PANG; Sai-Juan CHEN

    2004-01-01

    The t(8;21)translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia.This transtocation creates a fusion between the acute myelogenous leukemia 1(AML1,a transcription factor)gene on chromosome 21 and the eight-twenty-one(ETO,a zinc finger nuclear protein)gene on chromosome 8,leading to the repression of certain AML 1 target genes.We cloned NHR3 domain coding fragment into vector pGEX-6p-1 using PCR and obtained recombinant plasmid pGEX-6p-1-NHR3,which can be induced to stably overexpress fusion protein in E.coli.Through the purification on GST affinity chromatography column and PreScission protease cleavage,a large amount of NHR3 protein with high purity was obtained.In order to avoid possible interference of some strong negative charged molecules,NHR3 protein was further purified by Mono Q anion exchange chromatography.The NHR3 crystals were grown with hanging drop/vapor diffusion method and the first crystals appeared after four weeks at 18 ℃in 0.2 M Tris-sodium citrate dihydrate,0.1 M sodium cacodylate,pH 6.5,and 30% iso-propanol(V/V).ESI mass spectrum showed that the molecular weight of this domain was in agreement with its primary structure sequence prediction,and circular dichroism spectral data(190-250 nm)showed that NHR3 had a well-defined secondary structure of 25.9% α-helix,23.2% random coil and 50.9% turn,which was consistent with GOV4 software prediction.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Trindade, Inês B.; Fonseca, Bruno M. [Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras (Portugal); Matias, Pedro M. [Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras (Portugal); Instituto de Biologia Experimental e Tecnológica (iBET), Apartado 12, 2780-901 Oeiras (Portugal); Louro, Ricardo O.; Moe, Elin, E-mail: elinmoe@itqb.unl.pt [Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras (Portugal)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki [Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India); Jeyakanthan, Jeyaraman [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Baba, Seiki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 (Japan); Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Kuramitsu, Seiki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 (Japan); Shiro, Yoshitsugu [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Sekar, Kanagaraj, E-mail: sekar@serc.iisc.ernet.in [Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India); Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012 (India); Yokoyama, Shigeyuki, E-mail: sekar@serc.iisc.ernet.in [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India)

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

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    Kraschnefski, Mark J.; Scott, Stacy A. [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia); Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von [Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010 (Australia); Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia)

    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.

  11. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of molybdopterin synthase from Thermus thermophilus HB8

    Energy Technology Data Exchange (ETDEWEB)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki [Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India); Jeyakanthan, Jeyaraman; Ohmori, Miwa; Agari, Kazuko; Kitamura, Yoshiaki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Baba, Seiki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 (Japan); Ebihara, Akio; Shinkai, Akeo [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Kuramitsu, Seiki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 (Japan); Shiro, Yoshitsugu [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Sekar, Kanagaraj, E-mail: sekar@physics.iisc.ernet.in [Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India); Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012 (India); Yokoyama, Shigeyuki, E-mail: sekar@physics.iisc.ernet.in [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Bioinformatics Centre (Centre of Excellence in Structural Biology and Biocomputing), Indian Institute of Science, Bangalore 560 012 (India)

    2007-04-01

    The molybdopterin synthase from T. thermophilus HB8 was cloned, expressed, purified and crystallized. The crystals belong to space group P2{sub 1} and diffracted to a resolution of 1.64 Å. Thermus thermophilus is a Gram-negative aerobic thermophilic eubacterium which can grow at temperatures ranging from 323 to 355 K. In addition to their importance in thermostability or adaptation strategies for survival at high temperatures, the thermostable enzymes in thermophilic organisms contribute to a wide range of biotechnological applications. The molybdenum cofactor in all three kingdoms consists of a tricyclic pyranopterin termed molybdopterin that bears the cis-dithiolene group responsible for molybdenum ligation. The crystals of molybdopterin synthase from T. thermophilus HB8 belong to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 33.94, b = 103.32, c = 59.59 Å, β = 101.3°. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.

  12. Cloning, high-level expression, purification and characterization of a ...

    African Journals Online (AJOL)

    Cloning, high-level expression, purification and characterization of a staphylokinase variant, SakøC, ... African Journal of Biotechnology ... Hence in this study, we reported the cloning, high-level expression, purification and characterization of ...

  13. PredPPCrys: accurate prediction of sequence cloning, protein production, purification and crystallization propensity from protein sequences using multi-step heterogeneous feature fusion and selection.

    Directory of Open Access Journals (Sweden)

    Huilin Wang

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Soo-Young [Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784 (Korea, Republic of); Kang, Beom Sik [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Kim, Myung Hee [Systems Microbiology Research Center, Korea Research Institute of Biosciences and Biotechnology, Yusung, Daejon 305-806 (Korea, Republic of); Kim, Kyung Jin, E-mail: kkj@postech.ac.kr [Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784 (Korea, Republic of)

    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.

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

    Science.gov (United States)

    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).

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

    Science.gov (United States)

    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%.

  17. Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1.

    Science.gov (United States)

    Kernstock, Stefan; Koch-Nolte, Friedrich; Mueller-Dieckmann, Jochen; Weiss, Manfred S; Mueller-Dieckmann, Christoph

    2009-05-01

    Human ADP-ribosylhydrolase 1 (hARH1, ADPRH) cleaves the glycosidic bond of ADP-ribose attached to an Arg residue of a protein. hARH1 has been cloned, expressed heterologously in Escherichia coli, purified and crystallized in complex with K(+) and ADP. The orthorhombic crystals contained one monomer per asymmetric unit, exhibited a solvent content of 43% and diffracted X-rays to a resolution of 1.9 A. A prerequisite for obtaining well diffracting crystals was the performance of X-ray fluorescence analysis on poorly diffracting apo hARH1 crystals, which revealed the presence of trace amounts of K(+) in the crystal. Adding K-ADP to the crystallization cocktail then resulted in a crystal of different morphology and with dramatically improved diffraction properties.

  18. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of the biosynthetic N-acetylornithine aminotransferases from Salmonella typhimurium and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Rajaram, V.; Prasad, K.; Ratna Prasuna, P.; Ramachandra, N.; Bharath, S. R. [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

    2006-10-01

    Acetylornithine aminotransferases, members of the type I subgroup II family of PLP-dependent enzymes, from S. typhimurium and E. coli have been cloned, overexpressed, purified and crystallized. Acetylornithine aminotransferase (AcOAT) is a type I pyridoxal 5′-phosphate-dependent enzyme catalyzing the conversion of N-acetylglutamic semialdehyde to N-acetylornithine in the presence of α-ketoglutarate, a step involved in arginine metabolism. In Escherichia coli, the biosynthetic AcOAT also catalyzes the conversion of N-succinyl-l-2-amino-6-oxopimelate to N-succinyl-l,l-diaminopimelate, one of the steps in lysine biosynthesis. It is closely related to ornithine aminotransferase. AcOAT was cloned from Salmonella typhimurium and E. coli, overexpressed in E. coli and purified using Ni–NTA affinity column chromatography. The enzymes crystallized in the presence of gabaculine. Crystals of E. coli AcOAT (eAcOAT) only diffracted X-rays to 3.5 Å and were twinned. The crystals of S. typhimurium AcOAT (sAcOAT) diffracted to 1.9 Å and had a dimer in the asymmetric unit. The structure of sAcOAT was solved by the molecular-replacement method.

  19. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Fuzhou [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Wang, Chao [Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore); National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610 (Singapore); Fu, Qinqin [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Zhang, Lian-hui [Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore); Gao, Yong-gui, E-mail: ygao@ntu.edu.sg [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore)

    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.

  20. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.

    Science.gov (United States)

    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.

  1. Cloning, expression, purification, crystallization and preliminary crystallographic analysis of pseudo death-effector domain of HIPPI, a molecular partner of Huntingtin-interacting protein HIP-1

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Manisha; Majumder, Pritha; Bhattacharyya, Nitai P.; Dattagupta, Jiban K.; Sen, Udayaditya, E-mail: udayaditya.sen@saha.ac.in [Structural Genomics Section, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700064 (India)

    2006-12-01

    A pseudo death-effector domain (pDED) of HIPPI, a partner of Huntingtin-interacting protein HIP1, has been cloned, overexpressed and crystallized. The crystals of pDED-HIPPI diffracted to 2.2 Å. The formation of a heterodimer between Huntingtin-interacting protein-1 (HIP-1) and its novel partner HIPPI (HIP-1 protein interactor) through their pseudo death-effector domains (pDEDs) is a key step that recruits caspase-8 and initiates apoptosis. This could be one of the pathways by which apoptosis is increased in Huntington’s disease (HD). A construct consisting of the pDED of HIPPI has been cloned and overexpressed as 6NH-tagged protein and purified by Ni–NTA affinity chromatography. Crystals of the pDED of HIPPI were grown in space group P4{sub 1}, with unit-cell parameters a = b = 77.42, c = 33.31 Å and a calculated Matthews coefficient of 1.88 Å{sup 3} Da{sup −1} (33% solvent content) with two molecules per asymmetric unit.

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

    Science.gov (United States)

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

    2014-06-01

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

  3. Cloning, expression, purification, crystallization and preliminary X-ray studies of a pyridoxine 5′-phosphate oxidase from Mycobacterium smegmatis

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Colin J., E-mail: colin.jackson@csiro.au; Taylor, Matthew C.; Tattersall, David B.; French, Nigel G. [CSIRO Entomology, Black Mountain, ACT 2601 (Australia); Carr, Paul D.; Ollis, David L. [Research School of Chemistry, Australian National University, ACT 0200 (Australia); Russell, Robyn J.; Oakeshott, John G. [CSIRO Entomology, Black Mountain, ACT 2601 (Australia)

    2008-05-01

    Good-quality crystals of selenomethionine-substituted Msmeg-3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 Å using synchrotron radiation. Pyridoxine 5′-phosphate oxidases (PNPOxs) are known to catalyse the terminal step in pyridoxal 5′-phosphate biosynthesis in a flavin mononucleotide-dependent manner in humans and Escherichia coli. Recent reports of a putative PNPOx from Mycobacterium tuberculosis, Rv1155, suggest that the cofactor or catalytic mechanism may differ in Mycobacterium species. To investigate this, a putative PNPOx from M. smegmatis, Msmeg-3380, has been cloned. This enzyme has been recombinantly expressed in E. coli and purified to homogeneity. Good-quality crystals of selenomethionine-substituted Msmeg-3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 Å using synchrotron radiation.

  4. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SpyCEP, a candidate antigen for a vaccine against Streptococcus pyogenes.

    Science.gov (United States)

    Abate, Francesca; Malito, Enrico; Falugi, Fabiana; Margarit Y Ros, Immaculada; Bottomley, Matthew James

    2013-10-01

    Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen against which an effective vaccine does not yet exist. The S. pyogenes protein SpyCEP (S. pyogenes cell-envelope proteinase) is a surface-exposed subtilisin-like serine protease of 1647 amino acids. In addition to its auto-protease activity, SpyCEP is capable of cleaving interleukin 8 and related chemokines, contributing to GAS immune-evasion strategies. SpyCEP is immunogenic and confers protection in animal models of GAS infections. In order to structurally characterize this promising vaccine candidate, several SpyCEP protein-expression constructs were designed, cloned, produced in Escherichia coli, purified by affinity chromatography and subjected to crystallization trials. Crystals of a selenomethionyl form of a near-full-length SpyCEP ectodomain were obtained. The crystals diffracted X-rays to 3.3 Å resolution and belonged to space group C2, with unit-cell parameters a=139.2, b=120.4, c=104.3 Å, β=111°.

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

    Science.gov (United States)

    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…

  6. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis

    Energy Technology Data Exchange (ETDEWEB)

    Subburaman, P.; Austin, B.P.; Shaw, G.X.; Waugh, D.S.; Ji, X. (NCI)

    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}.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Feifei [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China); Gao, Feng [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of (China); Li, Honglin [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China); Gong, Weimin [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of (China); Zhou, Lin, E-mail: gdtb-bg@vip.163.com [Center for Tuberculosis Control of Guangdong Province, Guangzhou, People’s Republic of (China); Bi, Lijun, E-mail: gdtb-bg@vip.163.com [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China)

    2014-07-23

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

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Purification of 5-hydroxymethylfurfural (hmf) by crystallization

    DEFF Research Database (Denmark)

    2013-01-01

    This invention relates to an efficient procedure for purifying HMF by crystallization at low temperature from an organic solvent.......This invention relates to an efficient procedure for purifying HMF by crystallization at low temperature from an organic solvent....

  10. Purification of a Multidrug Resistance Transporter for Crystallization Studies

    Directory of Open Access Journals (Sweden)

    Kamela O. Alegre

    2015-03-01

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

  11. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    Directory of Open Access Journals (Sweden)

    Lu Jia

    2011-10-01

    Full Text Available Abstract Background Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Results Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

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

    Science.gov (United States)

    Hekmat, Dariusch

    2015-07-01

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

  13. Cloning, expression, and purification of glutamine synthetase from Clostridum acetobutylicum

    Energy Technology Data Exchange (ETDEWEB)

    Usdin, K.P.; Zappe, H.; Jones, D.T.; Woods, D.R.

    1986-09-01

    A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg/sup 2 +/ in the ..gamma..-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.

  14. Barley coleoptile peroxidases. Purification, molecular cloning, and induction by pathogens

    DEFF Research Database (Denmark)

    Kristensen, B.K.; Bloch, H.; Rasmussen, Søren Kjærsgård

    1999-01-01

    A cDNA clone encoding the Prx7 peroxidase from barley (Hordeum vulgare L.) predicted a 341-amino acid protein with a molecular weight of 36,515. N- and C-terminal putative signal peptides were present, suggesting a vacuolar location of the peroxidase. Immunoblotting and reverse-transcriptase poly...

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Kevin C., E-mail: kqian@rdg.boehringer-ingelheim.com [Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals Inc., Research and Development, 175 Briar Ridge Road, Ridgefield, CT 06877 (United States); Studts, Joey; Wang, Lian; Barringer, Kevin; Kronkaitis, Anthony; Peng, Charline; Baptiste, Alistair; LaFrance, Roger; Mische, Sheenah [Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals Inc., Research and Development, 175 Briar Ridge Road, Ridgefield, CT 06877 (United States); Farmer, Bennett [Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals Inc., Research and Development, 175 Briar Ridge Road, Ridgefield, CT 06877 (United States)

    2005-01-01

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

  17. Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB

    Energy Technology Data Exchange (ETDEWEB)

    Roujeinikova, Anna, E-mail: anna.roujeinikova@manchester.ac.uk [Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom)

    2008-04-01

    The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a putative peptidoglycan-binding domain of H. pylori MotB, a stator component of the bacterial flagellar motor, are reported. The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 Å, β = 112.5°. The crystals diffract X-rays to at least 1.6 Å resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 Å showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.

  18. Purification and crystallization of Kokobera virus helicase

    Energy Technology Data Exchange (ETDEWEB)

    De Colibus, Luigi; Speroni, Silvia [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy); Coutard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Forrester, Naomi L.; Gould, Ernest [Centre for Ecology and Hydrology (formerly Institute of Virology), Mansfield Road, Oxford OX1 3SR (United Kingdom); Canard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Mattevi, Andrea, E-mail: mattevi@ipvgen.unipv.it [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy)

    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.

  19. Identification, cloning, and purification of protein antigens of Treponema pallidum.

    Science.gov (United States)

    Stamm, L V; Dallas, W S; Ray, P H; Bassford, P J

    1988-01-01

    Difficulties in culturing the bacterium Treponema pallidum have greatly hindered syphilis research. In recent years, several laboratories have begun applying recombinant DNA technology to the study of this organism. Recent work is summarized concerning the expression of T. pallidum DNA in Escherichia coli. A number of E. coli clones expressing treponemal protein antigens have been identified. In one instance, a recombinant protein was purified to homogeneity and shown to be identical to a highly immunogenic, native T. pallidum membrane protein of molecular weight 39,000, which was designated the basic membrane protein (BMP) of this organism. In addition, recent experiments are described that were designed to identify cell-surface proteins that would serve as the primary focus of our cloning efforts. Results obtained with use of several different approaches strongly suggest that the outer membrane of T. pallidum is an antigenically inert structure largely devoid of protein. However, a class of low-molecular-weight protein antigens have been identified that are actively secreted into the extracellular medium. Attempts currently are being made to clone these secreted proteins and investigate their roles in the pathogenesis and immunobiology of syphilis.

  20. Cloning, expression, purification and characterization of tryptophan hydroxylase variants

    DEFF Research Database (Denmark)

    Boesen, Jane

    ). The main goal was to purify full-length hTPH1. Based on earlier results, hTPH1 was purified using detergent in the purification methods. After incubation of the hTPH1 sample with 0.1 % of n-dodecyl-β-D-maltopyranoside (DDM) the protein binds to the anion exchange column and elutes over a large area...... in the anion exchange, indicating that the protein still exists in different oligomer forms. This was also observed in the gel filtration. Variants of both hTPH1 and hTPH2 containing the regulatory domain or parts of it were constructed and tested for expression in Escherichia coli as well as solubility...... was determined and compared with parameters of chTPH2. Large differences were observed between the two isoforms and tryptophan inhibition was observed for chTPH1 but not for chTPH2. Mass spectrometric analysis of chTPH1 shows that the sample contains two species: chTPH1 and another species, which could...

  1. Research and Development of Crystal Purification for Product of Uranium Crystallization Process

    Energy Technology Data Exchange (ETDEWEB)

    Yano, K. [Japan Atomic Energy Agency - JAEA (Japan)

    2009-06-15

    Uranium crystallization has been developed as a part of advanced aqueous reprocessing for FBR spent fuel. Although the purity of uranyl nitrate hexahydrate (UNH) crystal from the crystallization process is supposed to meet a specification of FBR blanket fuel, an improvement of its purity is able to reduce the cost of fuel fabrication and storage (in case interim storage of recovered uranium is required). In this work, UNH crystal purification was developed as additional process after crystallization. Contamination of the crystal is caused by mother solution and solid state impurities. They are inseparable by washing and filtration. Mother solution on the surface of UNH crystals is removable by washing, but it is difficult to remove that in an obstructed part of crystalline aggregate by washing. Major elements of solid state impurities are cesium and barium. Cesium precipitates with tetravalent plutonium as a double nitrate, Cs{sub 2}Pu(NO{sub 3}){sub 6}. Barium crystallizes as Ba(NO{sub 3}){sub 2} because of its low solubility in nitric acid solution. It is difficult to separate their particle from UNH crystal by solid-liquid separation such as simple filtration. As a kind of crystal purification, there are some methods using sweating. Sweating is a phenomenon that a crystal melts partly below its melting point and it is caused by depression of freezing point due to impurity. It is considerably applicable for removal of mother solution. Concerning the solid state impurities, which has higher melting point than that of UNH crystal, it is supposed that they are separable by melting UNH crystal and filtration. The behaviors of impurities and applicability of sweating and melting-filtration operations to the purification for UNH crystal were investigated experimentally on a beaker and an engineering scale. With regard to behaviors of impurities, the conditions of cesium and barium precipitation were surveyed and it was clarified that there were most impurities on the

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

    Science.gov (United States)

    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…

  3. Phosphoric acid purification by suspension melt crystallization: Parametric study of the crystallization and sweating steps

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Baoming; Li, Jun; Qi, Yabing; Jia, Xuhong; Luo, Jianhong [Department of Chemical Engineering, Sichuan University, Chengdu 610065 (China)

    2012-10-15

    In order to purify phosphoric acid, the suspension melt crystallization process was studied. The suspension crystallization experiments were carried out with 80, 84 and 88 wt% phosphoric acid melt at the cooling rates of 0.05, 0.1 and 0.2 K/min, respectively. Sweating experiments were executed for various crystals obtained in suspension crystallization step. The purification effects of the sweating parameters including sweating time, initial inclusion amount and initial impurity content were studied. The inclusion fraction increases with the increase in cooling rate. The inclusion fraction of the crystals which were formed with feed concentration of 84 wt% phosphoric acid melt is lowest among the three feed concentrations. Different impurities have different purification performances during sweating. High inclusion amount and low impurity concentration favor the purification of H{sub 3}PO{sub 4}.0.5H{sub 2}O crystals during sweating. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  4. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    Directory of Open Access Journals (Sweden)

    Yvonne D Trigoso

    Full Text Available The enzyme dihydrodipicolinate reductase (DHDPR is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(PH dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3. The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

  5. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    Science.gov (United States)

    Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

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

    Energy Technology Data Exchange (ETDEWEB)

    Reyes-Grajeda, Juan Pablo [Unidad de Proteómica Médica, Instituto Nacional de Medicina Genómica, Mexico City (Mexico); Marín-García, Liliana [Instituto de Química, Universidad Nacional Autónoma de México (Mexico); Stojanoff, Vivian [Brookhaven National Laboratories, NSLS, Upton, New York (United States); Moreno, Abel, E-mail: carcamo@servidor.unam.mx [Instituto de Química, Universidad Nacional Autónoma de México (Mexico); Unidad de Proteómica Médica, Instituto Nacional de Medicina Genómica, Mexico City (Mexico)

    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.

  7. Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review.

    Science.gov (United States)

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2017-01-01

    The protein productions strategies are crucial towards the development of application based research and elucidating the novel purification strategies for industrial production. Currently, there are few innovative avenues are studies for cloning, upstream, and purification through efficient bioprocess development. Such strategies are beneficial for industries as well as proven to be vital for effectual therapeutic protein development. Though, these techniques are well documented, but, there is scope of addition to current knowledge with novel and new approaches and it will pave new avenues in production of recombinant microbial and non-microbial proteins including secondary metabolites. In this review, we have focussed on the recent development in clone selection, various modern fermentation and purification technologies and future directions in these emerging areas. Moreover, we have also highlighted notable perspectives and challenges involved in the bioengineering of such proteins, including quality by design, gene editing and pioneering ideas. The biopharmaceutical industries continue to shift towards more flexible, automated platforms and economical product development, which in turn can help in developing the cost effective processes and affordable drug development for a large community.

  8. Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review

    Directory of Open Access Journals (Sweden)

    Sanjeev K. Gupta

    2017-07-01

    Full Text Available The protein productions strategies are crucial towards the development of application based research and elucidating the novel purification strategies for industrial production. Currently, there are few innovative avenues are studies for cloning, upstream, and purification through efficient bioprocess development. Such strategies are beneficial for industries as well as proven to be vital for effectual therapeutic protein development. Though, these techniques are well documented, but, there is scope of addition to current knowledge with novel and new approaches and it will pave new avenues in production of recombinant microbial and non-microbial proteins including secondary metabolites. In this review, we have focussed on the recent development in clone selection, various modern fermentation and purification technologies and future directions in these emerging areas. Moreover, we have also highlighted notable perspectives and challenges involved in the bioengineering of such proteins, including quality by design, gene editing and pioneering ideas. The biopharmaceutical industries continue to shift towards more flexible, automated platforms and economical product development, which in turn can help in developing the cost effective processes and affordable drug development for a large community.

  9. Cloning, purification, crystallization and 1.57 Å resolution X-ray data analysis of AmsI, the tyrosine phosphatase controlling amylovoran biosynthesis in the plant pathogen Erwinia amylovora.

    Science.gov (United States)

    Benini, Stefano; Caputi, Lorenzo; Cianci, Michele

    2014-12-01

    The Gram-negative bacterium Erwinia amylovora is a destructive pathogen of plants belonging to the Rosaceae family. Amongst its pathogenicity factors, E. amylovora produces the exopolysaccharide amylovoran, which contributes to the occlusion of plant vessels, causing wilting of shoots and eventually resulting in plant death. Amylovoran biosynthesis requires the presence of 12 genes (from amsA to amsL) clustered in the ams region of the E. amylovora genome. They mostly encode glycosyl transferases (AmsG, AmsB, AmsD, AmsE, AmsJ and AmsK), proteins involved in amylovoran translocation and assembly (AmsH, AmsL and AmsC), and also a tyrosine kinase (AmsA) and a tyrosine phosphatase (AmsI), which are both involved in the regulation of amylovoran biosynthesis. The low-molecular-weight protein tyrosine phosphatase AmsI was overexpressed as a His6-tagged protein in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to a maximum resolution of 1.57 Å in space group P3121.

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Pathuri, Puja; Nguyen, Emily Tam [Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697 (United States); Luecke, Hartmut, E-mail: hudel@uci.edu [Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697 (United States); Department of Physiology and Biophysics, University of California, Irvine, CA 92697 (United States); Department of Information and Computer Sciences, University of California, Irvine, CA 92697 (United States)

    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.

  12. Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

    Science.gov (United States)

    He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

    2015-10-01

    An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories.

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

    Energy Technology Data Exchange (ETDEWEB)

    Perez, Zhanita N.; Musingarimi, Primrose [Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD (United States); Craig, Nancy L. [Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD (United States); Dyda, Fred; Hickman, Alison Burgess, E-mail: ahickman@helix.nih.gov [Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD (United States)

    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.

  14. Molecular cloning, expression, purification and crystallographic analysis of PRRSV 3CL protease

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Xinsheng; Feng, Youjun [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Graduate University, Chinese Academy of Sciences, Beijing 100049 (China); Zhao, Tiezhu [China Animal Disease Control Center, Beijing 100094 (China); Peng, Hao; Yan, Jinghua [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Qi, Jianxun [Graduate University, Chinese Academy of Sciences, Beijing 100049 (China); Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Jiang, Fan [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Tian, Kegong, E-mail: tiankg@263.net [China Animal Disease Control Center, Beijing 100094 (China); Gao, Feng, E-mail: tiankg@263.net [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); College of Biological Sciences, China Agricultural University, Beijing 100094 (China)

    2007-08-01

    Recombinant PRRSV 3CL protease was crystallized and the crystals diffracted to 2.1 Å resolution. 3CL protease, a viral chymotrypsin-like proteolytic enzyme, plays a pivotal role in the transcription and replication machinery of many RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the full-length 3CL protease from PRRSV was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that diffracted to 2.1 Å resolution and belong to space group C2, with unit-cell parameters a = 112.31, b = 48.34, c = 42.88 Å, β = 109.83°. The Matthews coefficient and the solvent content were calculated to be 2.49 Å{sup 3} Da{sup −1} and 50.61%, respectively, for one molecule in the asymmetric unit.

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

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, Department of Biochemistry, University of Leicester, Leicester LE1 9HN (United Kingdom)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Garrote, Ana M.; Redondo, Pilar [Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid (Spain); Montoya, Guillermo, E-mail: gmontoya@cnio.es [Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Muñoz, Inés G., E-mail: gmontoya@cnio.es [Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid (Spain)

    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%.

  17. Cloning, bacterial expression and crystallization of Fv antibody fragments

    Science.gov (United States)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  18. Influence of zone purification process on TlBr crystals for radiation detector fabrication

    Energy Technology Data Exchange (ETDEWEB)

    Hitomi, Keitaro [Department of Electronics, Tohoku Institute of Technology, 35-1 Yagiyama Kasumi-cho, Taihaku-ku, Sendai 982-8577 (Japan)], E-mail: hitomi@tohtech.ac.jp; Onodera, Toshiyuki; Shoji, Tadayoshi [Department of Electronics, Tohoku Institute of Technology, 35-1 Yagiyama Kasumi-cho, Taihaku-ku, Sendai 982-8577 (Japan)

    2007-08-21

    Thallium bromide (TlBr) is a wide gap compound semiconductor and is a promising material for fabrication of nuclear radiation detectors. In this study, the conventional zone refining method was employed to reduce the concentration of impurities in the TlBr crystals. In order to evaluate the efficiency of the zone purification, the zone purification process was repeated up to 300 times. The resistivity, the charge transport properties, and the spectroscopic performance of TlBr detectors fabricated from the crystals zone purified 1 time, 100 times, and 300 times were compared in this study in order to clarify the effectiveness of the zone purification process.

  19. Cloning, expression, purification and refolding of microtubule affinity-regulating kinase 4 expressed in Escherichia coli.

    Science.gov (United States)

    Naz, Farha; Asad, Mohd; Malhotra, Pawan; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2014-03-01

    Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a member of the family Ser/Thr kinase and involved in numerous biological functions including microtubule bundle formation, nervous system development, positive regulation of programmed cell death, cell cycle control, cell polarity determination, cell shape alterations, cell division etc. For various biophysical and structural studies, we need this protein in adequate quantity. In this paper, we report a novel cloning strategy for MARK4. We have cloned MARK4 catalytic domain including 59 N-terminal extra residues with unknown function and catalytic domain alone in PQE30 vector. The recombinant MARK4 was expressed in the inclusion bodies in M15 cells. The inclusion bodies were solubilized effectively with 1.5% N-lauroylsarcosine in alkaline buffer and subsequently purified using Ni-NTA affinity chromatography in a single step with high purity and good concentration. Purity of protein was checked on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by using mass spectrometry immunoblotting. Refolding of the recombinant protein was validated by ATPase assay. Our purification procedure is quick, simple and produces adequate quantity of proteins with high purity in a limited step.

  20. Human granulocyte colony stimulating factor (hG-CSF: cloning, overexpression, purification and characterization

    Directory of Open Access Journals (Sweden)

    Vanz Ana LS

    2008-04-01

    Full Text Available Abstract Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3 host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4% to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost

  1. Cloning, Expression and Purification of an Acetoacetyl CoA Thiolase from Sunflower Cotyledon

    Directory of Open Access Journals (Sweden)

    James H. Dyer, Anthony Maina, Iris D. Gomez, Melissa Cadet, Silke Oeljeklaus, Anke C. Schiedel

    2009-01-01

    Full Text Available Thiolase I and II coexist as part of the glyoxysomal β-oxidation system in sunflower (Helianthus annuus L. cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively. Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT.

  2. Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    L. Avilán

    1997-12-01

    Full Text Available We cloned the streptokinase (STK gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2013-09-01

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

  6. Separation and PurificationHomologous Compounds byof Dodecanedioic Acid from Its Falling Film Crystallization

    Institute of Scientific and Technical Information of China (English)

    李裕; 刘有智; 齐雪琴

    2004-01-01

    Separation and purification of dodecanedioic acid (DDDA) from its homologous compounds were studied experimentally by falling film crystallization (FFC). The influences of various operation parameters, including crystallizing time, flow rate of melt and temperature of glycerine bath, on purity of DDDA and crystallizing rate were investigated. Over 99% (by mole) DDDA was obtained for a feed composition of 96% (by mole). The main factors affecting the separation efficiency are flow rate of melt and temperature of glycerine bath. The crystallizing layer of DDDA was further purified by sweating and blasting. A set of optimized operation data are provided for better understanding the mechanism of heat and mass transfer in FFC, and for further industrial application of DDDA purification process.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2001-12-01

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

  9. Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

    DEFF Research Database (Denmark)

    Guerra, B; Niefind, K; Pinna, L A

    1998-01-01

    The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameter...

  10. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.

    Science.gov (United States)

    Iqbal, Irfana; Aftab, Muhammad Nauman; Afzal, Mohammed; Ur-Rehman, Asad; Aftab, Saima; Zafar, Asma; Ud-Din, Zia; Khuharo, Ateeque Rahman; Iqbal, Jawad; Ul-Haq, Ikram

    2015-02-01

    Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

  11. Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.

    Directory of Open Access Journals (Sweden)

    Marion Giffard

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Burgess, Benjamin R.; Dobson, Renwick C. J., E-mail: rdobson@unimelb.edu.au; Dogovski, Con [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Jameson, Geoffrey B. [Centre for Structural Biology, Institute of Fundamental Sciences, Massey University, Palmerston North (New Zealand); Parker, Michael W. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, Parkville, Victoria 3010 (Australia); St Vincents Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065 (Australia); Perugini, Matthew A., E-mail: rdobson@unimelb.edu.au [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, Parkville, Victoria 3010 (Australia)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, University of Leicester, Leicester LE1 9HN (United Kingdom)

    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.

  14. Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target

    OpenAIRE

    Soleimani, Neda; Mohabati Mobarez, Ashraf; Farhangi, Baharak

    2016-01-01

    Purpose Helicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori–specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid c...

  15. Effects of purification on the crystallization of lysozyme

    Science.gov (United States)

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

    1996-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Dhavala, Prathusha [Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku 20521 (Finland); Krasotkina, Julya [Institute for Biomedical Sciences, Russian Academy of Medical Sciences, Moscow (Russian Federation); Dubreuil, Christine; Papageorgiou, Anastassios C., E-mail: tassos.papageorgiou@btk.fi [Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku 20521 (Finland)

    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.

  17. Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target.

    Science.gov (United States)

    Soleimani, Neda; Mohabati Mobarez, Ashraf; Farhangi, Baharak

    2016-01-01

    Helicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori-specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid chromatography (FPLC) as a vaccination target. The hpaA gene was inserted into pET28a (+) as cloning and expression vectors respectively. The recombinant plasmid (pET-hpaA) was subjected to sequencing other than polymerase chain reaction (PCR) and digestion analysis. Protein expression was induced by adding 1 mM isopropyl-β-D-thiogalactoside to cultures of E. coli strain BL21 transformed with pET-hpaA. Protein expression assessed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein purification of flagellar sheath adhesion was by FPLC. The restriction endonuclease digestion, PCR amplification analysis showed that the hpaA gene of 730 bp was amplified from H. pylori DNA and sequencing analysis of the pET-hpaA confirmed the cloning accuracy and in frame insertion of hpaA fragment. SDS-PAGE analysis showed the expression of an approximately 29,000 Da protein. Sequencing results along with SDS-PAGE analysis confirms the expression of recombinant hpaA in the heterologous E. coli BL21. Conclusion A prokaryotic expression system for hpaA gene was successfully constructed. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.

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

    Energy Technology Data Exchange (ETDEWEB)

    Dobson, Renwick C. J., E-mail: rdobson@unimelb.edu.au; Atkinson, Sarah C. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, Victoria 3010 (Australia); Gorman, Michael A. [St Vincents Institute, 9 Princes Street, Fitzroy, Victoria 3065 (Australia); Newman, Janet M. [CSIRO Division of Molecular and Health Technologies, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Parker, Michael W. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, Victoria 3010 (Australia); St Vincents Institute, 9 Princes Street, Fitzroy, Victoria 3065 (Australia); Perugini, Matthew A. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, Victoria 3010 (Australia)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Quirk, Stephen; Seley-Radtke, Katherine L., E-mail: kseley@umbc.edu [Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Chemistry 405C, Baltimore, MD 21250 (United States)

    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.

  20. Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host.

    Science.gov (United States)

    Celińska, Ewelina; Białas, Wojciech; Borkowska, Monika; Grajek, Włodzimierz

    2015-03-01

    Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak activity (81 AU/L) was reached after only 29 h of culturing in 5-L bioreactors. Through simple purification procedure of ammonium sulfate precipitation and affinity chromatography, it was possible to purify the enzyme to apparent homogeneity (25-fold purification factor, at 5 % yield). The optimal conditions for the α-amylase activity were pH 5.0 and a temperature of 40 °C. The α-amylase studied here did not show any obligate requirement for Ca(2+) ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice.

  1. Cloning, expression, crystallization and preliminary X-ray analysis of the first two Ig domains from human Roundabout 1 (Robo1)

    Energy Technology Data Exchange (ETDEWEB)

    Morlot, Cecile [European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble (France); Hemrika, Wieger; Romijn, Roland A. [ABC Expression Center, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Gros, Piet [Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Cusack, Stephen, E-mail: cusack@embl.fr; McCarthy, Andrew A., E-mail: cusack@embl.fr [European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble (France)

    2007-08-01

    Robo1 is a large transmembrane receptor expressed on the axon growth cone. Activation of Robo1 by Slit proteins results in axon repulsion from the midline. Here, the purification and crystallization conditions of first two Ig domains of Robo1 are reported. Activation of Roundabout 1 (Robo1) by Slit proteins results in axon repulsion from the midline. Robo1 is a large transmembrane receptor expressed on the axon growth cone and the minimal Robo1-binding region required for Slit activation has been mapped to the N-terminal Ig1–2 domains. The cDNA encoding the first two Ig domains of Robo1 has been cloned and the protein has been expressed in HEK293 EBNA-1 mammalian cells. Here, the purification and crystallization conditions of this Robo1 construct are reported. The crystals are orthorhombic, space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 38.8, b = 69.4, c = 103.3 Å and one molecule in the asymmetric unit. X-ray diffraction data have been collected to 2.8 Å resolution on beamline ID29 at the ESRF.

  2. Potato virus X isolation and purification and coat protein gene cloning

    Directory of Open Access Journals (Sweden)

    L. Duplat

    2011-12-01

    Full Text Available In this work we describe the construction of potato virus X coat protein (PVX-CP gene clones from a Colombian isolate. Virus was isolated from field potato plants cultured at páramo de San Jorge. PVX particles were purified from Nicotiana tabacum leaves infected with a local lesion taken from Datura stramonium plants. The genomic RNA present in the virus particles consisted of a single species of about 6 Kb. cDNA synthesis representing genomic RNA was followed by Digoxigenin incorporation after priming with oligonucleotide 4DT/KPN. PCR amplification of CP gene sequence was made by using oligonucleotides OX6 and L/Kpn. An expected fragment of 870 bp, besides some 600-123 bp fragments, was obtained after PCR. The blunt-ended fragments were clonned into the PCR cloning pMOSblue plasmid. The insert size of the selected recombinant cDNA clones was determined by restriction analysis of the plasmidDNAwith Sma I and Hind III enzymes. Positive clones were also screened by direct colony PCR screening. The selected recombinant cDNA clones were stored in glycerol at .70 °C.

  3. Purification, characterization and molecular cloning of glycosylphosphatidylinositol-anchored arginine-specific ADP-ribosyltransferases from chicken.

    Science.gov (United States)

    Terashima, Masaharu; Osago, Harumi; Hara, Nobumasa; Tanigawa, Yoshinori; Shimoyama, Makoto; Tsuchiya, Mikako

    2005-08-01

    Mono-ADP-ribosylation is a post-translational modification that regulates the functions of target proteins or peptides by attaching an ADP-ribose moiety. Here we report the purification, molecular cloning, characterization and tissue-specific distribution of novel arginine-specific Arts (ADP-ribosyltransferases) from chicken. Arts were detected in various chicken tissues as GPI (glycosylphosphatidylinositol)-anchored forms, and purified from the lung membrane fraction. By molecular cloning based on the partial amino acid sequence using 5'- and 3'-RACE (rapid amplification of cDNA ends), two full-length cDNAs of chicken GPI-anchored Arts, cgArt1 (chicken GPI-anchored Art1) and cgArt2, were obtained. The cDNA of cgArt1 encoded a novel polypeptide of 298 amino acids which shows a high degree of identity with cgArt2 (82.9%), Art6.1 (50.2%) and rabbit Art1 (42.1%). In contrast, the nucleotide sequence of cgArt2 was identical with that of Art7 cloned previously from chicken erythroblasts. cgArt1 and cgArt2 proteins expressed in DT40 cells were shown to be GPI-anchored Arts with a molecular mass of 45 kDa, and these Arts showed different enzymatic properties from the soluble chicken Art, Art6.1. RNase protection assays and real-time quantitative PCR revealed distinct expression patterns of the two Arts; cgArt1 was expressed predominantly in the lung, spleen and bone marrow, followed by the heart, kidney and muscle, while cgArt2 was expressed only in the heart and skeletal muscle. Thus GPI-anchored Arts encoded by the genes cgArt1 and cgArt2 are expressed extensively in chicken tissues. It may be worthwhile determining the functional roles of ADP-ribosylation in each tissue.

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

    Directory of Open Access Journals (Sweden)

    Ho-Hsien Lee

    2014-09-01

    Full Text Available 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.

  5. Cloning

    Science.gov (United States)

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  6. Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

    CERN Document Server

    Foulon, V; Croes, K; Waelkens, E

    1999-01-01

    Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

  7. Purification and crystallization of yeast hexokinase isoenzymes. Characterization of different forms by chromatofocusing.

    Science.gov (United States)

    Jacob, L; Beecken, V; Bartunik, L J; Rose, M; Bartunik, H D

    1991-11-29

    The yeast hexokinase isoenzymes PI and PII have been purified in large amounts (20 mg) from overproducing yeast strains. The purification procedures of hexokinase PI and PII include anion-exchange chromatography on DEAE-Sephacel and chromatofocusing on PBE 94, hydrophobic interaction chromatography on phenyl-Sepharose (necessary for the isolation of the isoenzyme PI); in the final step either a Mono Q HR 5/5 or a Fractogel EMD TMAE 650(S) column was used. Hexokinase preparations were characterized before crystallization by chromatofocusing on a Mono P HR 5/20 FPLC column, where different forms of hexokinase can be rapidly distinguished by their elution behaviour. From both purified hexokinase PI and PII, large crystals were grown that diffract X-rays to high resolution.

  8. Isolation, Cloning, Expression and Purification of Recombinant RhD Antigen from Cord Blood

    Directory of Open Access Journals (Sweden)

    M Habibi Roudkenar

    2008-09-01

    Full Text Available "nBackground: Rh (Rhesus is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blod, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study."nMethods: Total RNAs were extracted from cord blood (O+.  The quality of RNA was determined by electrophoresis. In or­der to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT-PCR. The iso­lated RhD gene was   cloned to pUC18 vector and transformed to DH5α. The confirmed construct was sub cloned into expres­sion vector, pBADgIII/A, and expressed in Top10 E.coli. The expressed protein was characterized by SDS-PAGE and western blot analysis. Antigenicity of the expressed protein was assessed by ELISA using commercially available hu­man anti-RhD polyclonal   antibody with   peroxidase conjugated goat anti-human IgG, IgM, IgA as secondary antibody. "nRe­sults: RhD gene was successfully cloned and expressed. The expected size of recombinant RhD protein was detected in SDS-PAGE, and confirmed by dot and western blot analysis. RhD antibody reacted with recombinant RhD antigen as well as with RhD polypeptide extracted from RBCs membrane."nConclusion: The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein and could be applica­ble for production anti- D antibody in an animal model.

  9. Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein

    Directory of Open Access Journals (Sweden)

    Ali Dina

    2016-01-01

    Full Text Available In Leishmania species, protein disulfide isomerase (PDI is an essential enzyme that catalyzes thiol-disulfide interchange. The present work describes the isolation, cloning, sequencing and expression of the pdI-2 gene. Initially, the gene was amplified from L. tropica genomic DNA by PCR using specific primers before cloning into the expression vector pET-15b. The construct pET/pdI-2 was transformed into BL21(DE3 cells and induced for the protein expression. SDS-PAGE and western blot analysis showed that the expressed protein is about 51 kDa. Cloned gene sequence analysis revealed that the deduced amino acid sequence showed significant homology with those of several parasites PDIs. Finally, recombinant protein was purified with a metal-chelating affinity column. The putative protein was confirmed as a thiol - disulfide oxidoreductase by detecting its activity in an oxidoreductase assay. Assay result of assay suggested that the PDI-2 protein is required for both oxidation and reduction of disulfide bonds in vitro. Antibodies reactive with this 51 kDa protein were detected by Western blot analysis in sera from human infected with L. tropica. This work describes for the first time the enzymatic activity of recombinant L. tropica PDI-2 protein and suggests a role for this protein as an antigen for the detection of leishmaniasis infection.

  10. Expression, purification and crystallization of an atypical class C acid phosphatase from Mycoplasma bovis.

    Science.gov (United States)

    Singh, Harkewal; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2011-10-01

    Class C acid phosphatases (CCAPs) are 25-30 kDa bacterial surface proteins that are thought to function as broad-specificity 5',3'-nucleotidases. Analysis of the newly published complete genome sequence of Mycoplasma bovis PG45 revealed a putative CCAP with a molecular weight of 49.9 kDa. The expression, purification and crystallization of this new family member are described here. Standard purification procedures involving immobilized metal-ion affinity chromatography and ion-exchange chromatography yielded highly pure and crystallizable protein. Crystals were grown in sitting drops at room temperature in the presence of PEG 3350 and HEPES buffer pH 7.5 and diffracted to 2.3 Å resolution. Analysis of diffraction data suggested a primitive monoclinic space group, with unit-cell parameters a = 78, b = 101, c = 180 Å, β = 92°. The asymmetric unit is predicted to contain six molecules, which are likely to be arranged as three dimers.

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

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Colin J.; Carr, Paul D.; Kim, Hye-Kyung; Liu, Jian-Wei; Ollis, David L., E-mail: ollis@rsc.anu.edu.au [The Research School of Chemistry, Australian National University, ACT 0200 (Australia)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Linke, Christian, E-mail: clin180@ec.auckland.ac.nz [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Caradoc-Davies, Tom T. [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Australian Synchrotron, Clayton, Victoria 3168 (Australia); Proft, Thomas [School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Baker, Edward N. [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand)

    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.

  13. Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

    DEFF Research Database (Denmark)

    Guerra, B; Niefind, K; Pinna, L A

    1998-01-01

    a = 142.6, b = 61.3, c = 45.6 A, beta = 103.3 degrees and diffract X-rays to at least 2.0 A resolution. The calculated crystal packing parameter is Vm = 2.47 A3 Da-1 suggesting that one CK2alpha molecule is contained in the asymmetric unit and that the solvent content of the unit cell is 50%.......The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameters...

  14. Purification, crystallization and preliminary X-ray analysis of uridine phosphorylase from Salmonella typhimurium.

    Science.gov (United States)

    Dontsova, Mariya V; Savochkina, Yulia A; Gabdoulkhakov, Azat G; Baidakov, Sergey N; Lyashenko, Andrey V; Zolotukhina, Maria; Errais Lopes, Liubov; Garber, Mariya B; Morgunova, Ekaterina Yu; Nikonov, Stanislav V; Mironov, Alexandr S; Ealick, Steven E; Mikhailov, Al 'Bert M

    2004-04-01

    The structural udp gene encoding uridine phosphorylase (UPh) was cloned from the Salmonella typhimurium chromosome and overexpressed in Escherichia coli cells. S. typhimurium UPh (StUPh) was purified to apparent homogeneity and crystallized. The primary structure of StUPh has high homology to the UPh from E. coli, but the enzymes differ substantially in substrate specificity and sensitivity to the polarity of the medium. Single crystals of StUPh were grown using hanging-drop vapor diffusion with PEG 8000 as the precipitant. X-ray diffraction data were collected to 2.9 A resolution. Preliminary analysis of the diffraction data indicated that the crystal belonged to space group P6(1(5)), with unit-cell parameters a = 92.3, c = 267.5 A. The solvent content is 37.7% assuming the presence of one StUPh hexamer per asymmetric unit.

  15. Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Kandasamy Eniyan; Urmi Bajpai

    2015-01-01

    Objective:To clone, express and purify2-methylcitrate synthase(Rv1131) gene of Mycobacterium tuberculosis(M. tuberculosis) and to study its structural characteristics using various bioinformatics tools.Methods:Rv1131 gene was amplified by polymerase chain reaction usingM. tuberculosisH37Rv genomicDNA and cloned into pGEM-T easy vector and sequenced. The gene was sub-cloned in pET28c vector, expressed inEscherichia coliBL21(E. coliBL21) (DE3) cells and the recombinant protein was identified byWestern blotting.The protein was purified usingNickel affinity chromatography and the structural characteristics like sub-cellular localization, presence of transmembrane helices and secondary structure of the protein were predicted by bioinformatics tools.Tertiary structure of the protein and phylogenetic analysis was also established byin silico analysis.Results:The expression of the recombinant protein (Rv1131) was confirmed by western blotting using anti-HIS antibodies and the protein was purified from the soluble fraction.In silicoanalysis showed that the protein contains no signal peptide and transmembrane helices.Active site prediction showed that the protein has histidine and aspartic acid residues at242,281 &332 positions respectively.Phylogenetic analysis showed 100% homology withmajor mycobacterial species.Secondary structure predicts2-methylcitrate synthase contain51.9% alpha-helix,8.7% extended strand and39.4% random coils.Tertiary structure of the protein was also established.Conclusions:The enzyme2-methylcitrate synthase from M. tuberculosisH37Rv has been successfully expressed and purified.The purified protein will further be utilized to develop assay methods for screening new inhibitors.

  16. cDNA cloning, prokaryotic expression and purification of rat α-synuclein

    Institute of Scientific and Technical Information of China (English)

    Xin LI; Yao-Hua LI; Jun-Yan HAN; Shun YU; Biao CHEN

    2006-01-01

    Objective To clone the cDNA of rat α-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat α-Syn protein. Methods Rat α-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotie expressing vector. The recombinant plasmid containing rat α-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat α-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat α-Syn protein. The recombinant rat α-Syn protein was further purified using Superdex S200 gel filtration.Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat α-Syn. After transformation, the recombinant plasmid pGEX-raSyn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat α-Syn.Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against α-Syn. Conclusion The rat α-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat α-Syn recombinant protein was produced.

  17. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein.

    Science.gov (United States)

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Kim, Suk

    2016-03-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-01

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

  19. Expression, purification, crystallization and preliminary crystallographic analysis of the proliferation-associated protein Ebp1

    Energy Technology Data Exchange (ETDEWEB)

    Kowalinski, Eva; Bange, Gert; Wild, Klemens; Sinning, Irmgard, E-mail: irmi.sinning@bzh.uni-heidelberg.de [Heidelberg University Biochemistry Center, INF 328, D-69120 Heidelberg (Germany)

    2007-09-01

    Preliminary X-ray analysis of the proliferation-associated protein Ebp1 from Homo sapiens is provided. ErbB-3-binding protein 1 (Ebp1) is a member of the family of proliferation-associated 2G4 proteins (PA2G4s) and plays a role in cellular growth and differentiation. Ligand-induced activation of the transmembrane receptor ErbB3 leads to dissociation of Ebp1 from the receptor in a phosphorylation-dependent manner. The non-associated protein is involved in transcriptional and translational regulation in the cell. Here, the overexpression, purification, crystallization and preliminary crystallographic studies of Ebp1 from Homo sapiens are reported. Initially observed crystals were improved by serial seeding to single crystals suitable for data collection. The optimized crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2 and diffracted to a resolution of 1.6 Å.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide.

    Science.gov (United States)

    Fu, L; Hou, Y L; Ding, X; Du, Y J; Zhu, H Q; Zhang, N; Hou, W R

    2016-08-30

    The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

  2. Purification, Gene Cloning and Expression of an Acidic Phospholipase A2 from Agkistrodon shedaoensis Zhao

    Institute of Scientific and Technical Information of China (English)

    Qian JIN; Li-Xia YANG; Hao-Mang JIAO; Bin LU; Yu-Qun WU; Yuan-Cong ZHOU

    2004-01-01

    A protein with the activity of phospholipase A2 named asAPLA2 was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column,Source S and Mono Q FPLC. Its molecular weight was estimated as 19 kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1 μmol/L ADP, and the IC50 was determined to be 6 μmol/L. Degenerate primer was designed and synthesized according to the Nterminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares.The gene was then cloned into the expression plasmid pET-40b(+) and expressed in E. Coli BL21(DE3).Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the nativeenzyme.

  3. Purification, Gene Cloning and Expression of an Acidic Phospholipase A2 from Agkistrodon shedaoensis Zhao

    Institute of Scientific and Technical Information of China (English)

    QianJIN; Li-XiaYANG; Hao-MangJIAO; BinLU; Yu-QunWU; Yuan-CongZHOU

    2004-01-01

    A protein with the activity of phospholipase A2 named asAPLA2 was pmified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column,Source S and Mono Q FPLC. Its molecular weight was estimated as 19kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1μmol/L ADP, and the IC50 was determined to be 6μmol/L. Degenerate primer was designed and synthesized according to the Nterminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares.The gene was then cloned into the expression plasmid pET-40b(+) and expressed in E.coli BL21(DE3).Western blot analysis indicated that the expressed protein cross-reacted with the antibc dy against the native enzyme.

  4. Purification, biochemical characterization, and genetic cloning of the phytase produced by Burkholderia sp. strain a13.

    Science.gov (United States)

    Graminho, Eduardo Rezende; Takaya, Naoki; Nakamura, Akira; Hoshino, Takayuki

    2015-01-01

    A phytase-producing bacterium, Burkholderia sp. a13 (JCM 30421), was isolated from Lake Kasumigaura by enrichment cultivation using minimum medium containing phytic acid as the sole phosphorus source. The phytase production by strain a13 was induced by the presence of phytic acid and repressed by the addition of glucose. The purified enzyme had a molecular weight of 44 kDa and a phytase activity of 174 μmol min(-1) mg(-1). The enzyme showed broad substrate specificity, but the highest activity was observed with phytic acid. The enzyme activity was strongly inhibited by Cu(2+), Zn(2+), Hg(2+), and iodoacetic acid, indicating the requirement of a thiol group for the activity. Genetic cloning reveals that the mature portion of this enzyme consists of 428 amino acids with a calculated molecular weight of 46 kDa. The amino acid sequence showed the highest similarity to the phytase produced by Hafnia alvei with 48% identity; it also contained histidine acid phosphatase (HAP) motifs (RHGXRXP and HD), indicating the classification of this enzyme in the HAP phytase family. We have successfully expressed the cloned gene in Escherichia coli from its putative initiation codon, showing that the gene actually encodes the phytase.

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

    Science.gov (United States)

    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.

    Science.gov (United States)

    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. © 2012 The Authors Journal compilation © 2012 FEBS.

  7. Cloning, sequencing and functional expression of cytosolic malate dehydrogenase from Taenia solium: Purification and characterization of the recombinant enzyme.

    Science.gov (United States)

    Nava, Gabriela; Laclette, Juan P; Bobes, Raúl; Carrero, Julio C; Reyes-Vivas, Horacio; Enriquez-Flores, Sergio; Mendoza-Hernández, Guillermo; Plancarte, Agustín

    2011-07-01

    We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. K(cat) values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962s(-1), respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56Kb genomic contig assembly is also reported.

  8. (+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia. Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.

    Science.gov (United States)

    Dinkova-Kostova, A T; Gang, D R; Davin, L B; Bedgar, D L; Chu, A; Lewis, N G

    1996-11-15

    Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

  9. Cloning, expression, and purification of a synthetic human growth hormone in Escherichia coli using response surface methodology.

    Science.gov (United States)

    Zamani, Mozhdeh; Berenjian, Aydin; Hemmati, Shiva; Nezafat, Navid; Ghoshoon, Mohammad Bagher; Dabbagh, Fatemeh; Mohkam, Milad; Ghasemi, Younes

    2015-03-01

    The aim of this study was to achieve high-level production of the human growth hormone (hGH) in the prokaryotic expression system. In this regard, we performed cloning, expression, and purification of a synthetic hGH gene in BL21 (DE3) strain of E. coli. The hGH production was determined by SDS-PAGE and western blotting techniques, and then the protein concentration was determined by the Bradford assay. To gain insight into the effect of different nutrients on the growth of E. coli and hGH production, in a preliminary assessment nine different types of the basal medium were analyzed. The highest growth of E. coli and hGH production were observed in TB and SOB media. Accordingly, design of experiments was employed for screening the most significant nutrients, and central composite face design was applied for the optimization. The optimum medium consisted of yeast extract (10 g/L), tryptone (10 g/L), and K2HPO4 (2 g/L). The optimum hGH concentration was 391 mg/L, which was 3-fold higher than the hGH concentration in the LB basal medium (119 mg/L). This production rate is the highest hGH concentration reported in the IPTG-inducible expression systems.

  10. The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

    Science.gov (United States)

    Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinović-Carugo, Kristina; Pinotsis, Nikos

    2014-06-01

    Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

  11. Gene cloning, expression, purification and characterization of lipoprotein- associated phospholipase A2 in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Fu-junZHANG; Yi-pingWANG

    2005-01-01

    AIM To express and purify Lipoprotein -associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors through the recombinant Lp-PLA2. METHODS The full-length gene of Lp-PLA2 was cloned from the differentiated THP-1 cells by RT-PCR and PCR. The Lp-PLA2 gene was subcloned into the Pichia expression vector pPIC9 and introduced a sequence encoding a C-terminal stretch of six histidine residues at the same time. The recombinant plasmid was transformed into Pichia pastoris GS115 by spheroplasting and the gene was then integrated into the GS115 genome. Lp- PLA2 was expressed in the yeast strain GS115 by inducing with 0.5% methanol.

  12. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Directory of Open Access Journals (Sweden)

    Juhi Sikarwar

    2013-01-01

    Full Text Available Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3. The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA chromatography.

  13. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Science.gov (United States)

    Sikarwar, Juhi; Kaushik, Sanket; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

    2013-01-01

    Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk) has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3). The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography. PMID:23662205

  14. Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Brillowska-Dabrowska, Anna; Ahring, Birgitte Kiær

    2013-01-01

    Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis...... (14000 kU) of pure His6-tagged Tte AP (153 kU/mg) from 1 liter of culture. The optimal conditions of Tte AP endo-, exonuclease and 3'-nuclease activity were investigated using fluorescein labeled dsDNA with inserted AP sites and ssDNA. Optimal Tte AP endonuclease activity was observed at 70-75 degrees C...... DNA templates. Tte AP exonuclease eliminated AP-sites in DNA template and improved the efficiency of DNA amplification....

  15. Purification, crystallization and preliminary crystallographic investigations of selenium-containing phycocyanin from se-lenium-rich algae (Spirulina platensis)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The selenium-containing phycocyanin from the selenium-rich algae (Spirulina platensis) has been crystallized in two crystal forms by the hanging-drop vapor diffusion techniques. A chromatographic procedure of gel filtration and anion exchange was used for purification. Form I crystal with space group P21 and cell parameters a =108.0 ?, b = 117.0 ?, c = 184.0 ?, b = 90.2° and 12(ab) units in the asymmetric unit was obtained by using (NH4)2SO4 as precipitant. These crystals diffract up to 2.8 ?. Form II crystal obtained by using PEG4000 as precipitant belongs to space group P63 with unit cell constants a = 155.0 ?, c = 40.3 ?, g =120.0° and one(ab) unit in the asymmetric unit. The crystals diffract beyond 2.9 ?. The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.

  16. CLONING, PURIFICATION AND CHARACTERIZATION OF HALOTOLERANT XYLANASE FROM Geobacillus Thermodenitrificans C5

    Directory of Open Access Journals (Sweden)

    Muhammad Irfan

    2016-06-01

    Full Text Available High levels of extracellular xylanase activity (994.50 IU/ml produced by Geobacillus thermodenitrificans C5 originated gene was detected when it was expressed in E. coli BL21 host. Thermostable xylanase (GthC5Xyl was purified to homogeneity and showed a molecular mass of approximately 44 kDa according to SDS-PAGE. The specific activity of the purified GthC5Xyl was up to 1243.125IU/mg with a 9.89-fold purification. The activity of GthC5Xyl was stimulated by CoCl2, MnSO4, CuSO4, MnCl2 but was inhibited by FeSO4, Hg, CaSO4. GthC5Xyl showed resistant to SDS, Tween 20, Triton X-100, β- Mercaptoethanol, PMSF, DTT, NEM and DEPC, SDS, and EDTA. A greater affinity for oat spelt xylan was exhibited by GthC5Xyl with maximum enzymatic activity at 60°C and 6.0 pH. The activity portrayed by GthC5Xyl was found to be acellulytic with stability at high temperature (70°C-80°C and low pH (4.0 to 8.0. Xylobiose and xylopentose were the end products of proficient oat spelt xylanase hydrolysis by GthC5Xyl. High SDS resistance and broader stability of GthC5Xyl proves it to be worthy of impending application in numerous industrial processes especially textile, detergents and animal feed industry.

  17. Hyperentanglement purification and concentration assisted by diamond NV centers inside photonic crystal cavities

    Science.gov (United States)

    Ren, Bao-Cang; Deng, Fu-Guo

    2013-11-01

    Hyperentanglement has attracted much attention due to its fascinating applications in quantum communication. However, it is impossible to purify a pair of photon systems in a mixed hyperentangled state with errors in two degrees of freedom using linear optical elements only, far different from all the existing entanglement purification protocols in a degree of freedom (DOF) for quantum systems. Here, we investigate the possibility of purifying a spatial-polarization mixed hyperentangled Bell state with the errors in both the spatial-mode and polarization DOFs, resorting to the nonlinear optics of a nitrogen-vacancy (NV) center in a diamond embedded in a photonic crystal cavity coupled to a waveguide. We present the first hyperentanglement purification protocol for purifying a pair of two-photon systems in a mixed hyperentangled Bell state with the errors in two DOFs. We also propose an efficient hyperentanglement concentration protocol for a partially hyperentangled Bell pure state, which has the maximal success probability in principle. These two protocols are useful in long-distance quantum communication with hyperentanglement.

  18. Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

    Energy Technology Data Exchange (ETDEWEB)

    Pasquo, Alessandra [ENEA Casaccia Research Centre, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale, PO Box 2400, I-00100 Rome (Italy); Bonamore, Alessandra; Franceschini, Stefano; Macone, Alberto; Boffi, Alberto; Ilari, Andrea, E-mail: andrea.ilari@uniroma1.it [Istituto di Biologia e Patologia Molecolari, CNR (IBPM) and Department Of Biochemical Sciences, University of Roma ‘La Sapienza’, Piazza Aldo Moro 5, 00179 Roma (Italy); ENEA Casaccia Research Centre, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale, PO Box 2400, I-00100 Rome (Italy)

    2008-04-01

    The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction.

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

    Science.gov (United States)

    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.

  20. The Myb domain of LUX ARRHYTHMO in complex with DNA: expression, purification and crystallization.

    Science.gov (United States)

    Silva, Catarina S; Lai, Xuelei; Nanao, Max; Zubieta, Chloe

    2016-05-01

    LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock. Lux mutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein-DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using the MeshAndCollect workflow available at the ESRF.

  1. Cloning, Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells.

    Science.gov (United States)

    Shrestha, Pravesh; Yun, Ji-Hye; Kim, Woo Taek; Kim, Tae-Yoon; Lee, Weontae

    2016-03-01

    A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.

  2. Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

    Institute of Scientific and Technical Information of China (English)

    Haibo Wang; Duoduo Yuan; Rong Xu; Cheng-Wu Chi

    2011-01-01

    Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum, In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structurefunction studies.

  3. CLONING AND PURIFICATION OFA REPRESSOR PROTEIN FROM ARTHROBACTER NICOTINOVORANS PAO1

    Directory of Open Access Journals (Sweden)

    Roderich Brandsch

    2011-09-01

    Full Text Available The pAO1 megaplasmid of A. nicotinovorans consists of 165 ORF's related mainly to nicotine degradation, uptake and utilization of carbohydrates, amino acids and sarcosine The putative sugar catabolic pathway consists of 11 ORFś organized as a single operon and coding for an ABC-type sugar-transport system and several putative oxidoreductases and dehydrogenases. The current work is focused on orf32, a putative PdhR related protein, most probably involved in the control of the whole operon. The approx. 700 kb orf32 gene was cloned in the pH6EX3 plasmid vector and the gene product purified to homogeneity as a 29 kDa His-tagged recombinant protein. As indicated by GPC, it consists of a monomeric protein with a native molecular weight of 32 kDa. The specific UV/Vis spectra showed only a single peak at 280 nm common for all proteins and did not indicated the presence of any colored cofactors. This is in good agreement with the fact that PdhR-family proteins contain a winged helix-turn-helix (wHTH domain responsible for DNA binding, and not a Zn-finger or any other metal containing domains

  4. Purification and molecular cloning of a new galactose-specific lectin from Bauhinia variegata seeds

    Indian Academy of Sciences (India)

    Luciano S Pinto; Celso S Nagano; Taianá M Oliveira; Tales R Moura; Alexandre H Sampaio; Henri Debray; Vicente P Pinto; Odir A Dellagostin; Benildo S Cavada

    2008-09-01

    A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose–agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B. variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.

  5. Purification and molecular cloning of a new galactose-specific lectin from Bauhinia variegata seeds.

    Science.gov (United States)

    Pinto, Luciano S; Nagano, Celso S; Oliveira, Taianá M; Moura, Tales R; Sampaio, Alexandre H; Debray, Henri; Pinto, Vicente P; Dellagostin, Odir A; Cavada, Benildo S

    2008-09-01

    A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B.variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.

  6. Cloning, expression, and purification of a new antimicrobial peptide gene from Musca domestica larva.

    Science.gov (United States)

    Pei, Zhihua; Sun, Xiaoning; Tang, Yan; Wang, Kai; Gao, Yunhang; Ma, Hongxia

    2014-10-01

    Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram- bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.

  7. Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

    Science.gov (United States)

    Hanner, M; Moebius, F F; Flandorfer, A; Knaus, H G; Striessnig, J; Kempner, E; Glossmann, H

    1996-07-23

    Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone.

  8. Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

    Directory of Open Access Journals (Sweden)

    Tzeng Kuo-Ching

    2011-05-01

    Full Text Available Abstract Background Most isolates of Pectobacterium carotovorum subsp. carotovorum (Pcc produce bacteriocins. In this study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C. Results A carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in Escherichia coli DH5α after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa. Conclusion This study shown that another nuclease type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity of CaroS2I.

  9. Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

    Directory of Open Access Journals (Sweden)

    F.C. Cançado

    2008-11-01

    Full Text Available cDNA coding for two digestive lysozymes (MdL1 and MdL2 of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8 at low ionic strength (μ = 0.02, which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2. However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9 is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI indicate that MdL2 (pI = 6.7 is less positively charged than MdL1 (pI = 7.7 at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

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

    Energy Technology Data Exchange (ETDEWEB)

    Boeshans, Karen M. [X-ray Crystallography Facility, NIAMS, National Institutes of Health, Bethesda, MD 20892 (United States); Wolf, Ronald; Voscopoulos, Christopher [Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Gillette, William; Esposito, Dominic [Protein Expression Laboratory, Research Technology Program, National Cancer Institute, SAIC-Frederick Inc., Frederick, MD 21702 (United States); Mueser, Timothy C. [Department of Chemistry, University of Toledo, Toledo, OH 43606 (United States); Yuspa, Stuart H. [Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Ahvazi, Bijan, E-mail: ahvazib@mail.nih.gov [X-ray Crystallography Facility, NIAMS, National Institutes of Health, Bethesda, MD 20892 (United States)

    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.

  11. Cloning, expression, purification and characterization of patatin, a novel phospholipase A.

    Science.gov (United States)

    Hirschberg, H J; Simons, J W; Dekker, N; Egmond, M R

    2001-10-01

    Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.

  12. The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I.

    Science.gov (United States)

    Graciano, Luciana; Corrêa, Juliana Moço; Gandra, Rinaldo Ferreira; Seixas, Flavio Augusto Vicente; Kadowaki, Marina Kimiko; Sampaio, Silvio César; Silva, José Luis da Conceição; Osaku, Clarice Aoki; Simão, Rita de Cássia Garcia

    2012-09-01

    The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-L-Arabinofuranosidase (β-Xyl I-α-L-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-L-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-L-Ara exhibited a specific β-Xylosidase I activity of 1.25 U mg(-1) to oNPX and a specific α-L-Arabinofuranosidase activity of 0.47 U mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89 ± 0.13 mM and 1.4 ± 0.04 μM min(-1) to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.

  13. Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    Lina Lu

    2014-09-01

    Full Text Available The vacuolar (H+-ATPase (V-ATPase of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we cloned, expressed and purified the subunit H from midgut of Mythimna separata Walker. Through RACE (rapidly amplification of cDNA ends technique, we got 1807 bp full length of subunit H, and to keep the nature structure of subunit H, we constructed Baculovirus expression vector with His-tag in the C-terminal and expressed the recombinant protein in insect sf9 cells, thereafter, purified the recombinant protein by Ni-NTA columns. Results of SDS-PAGE, western blotting and mass spectrometry showed that the recombinant protein was successfully expressed. The method of expressing and purifying M. separata subunit H will provide a foundation for obtaining the crystal of subunit H and further study of the design of novel insecticides based on its structure and function.

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

    Science.gov (United States)

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

    2015-10-01

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

  15. Expression, Purification and Crystal Structure of a Truncated Acylpeptide Hydrolase from Aeropyrum pernix K1

    Institute of Scientific and Technical Information of China (English)

    Hai-Feng ZHANG; Bai-Song ZHENG; Ying PENG; Zhi-Yong LOU; Yan FENG; Zi-He RAO

    2005-01-01

    Acylpeptide hydrolase (APH) catalyzes the N-terminal hydrolysis of Nα-acylpeptides to release Nα-acylated amino acids. The crystal structure of recombinant APH from the thermophilic archaeon Aeropyrum pernix K1 (apAPH) was reported recently to be at a resolution of 2.1 A using X-ray diffraction. A truncated mutant of apAPH that lacks the first short α-helix at the N-terminal, apAPH-△(1-21), was cloned, expressed,characterized and crystallized. Data from biochemical experiments indicate that the optimum temperature of apAPH is decreased by 15 ℃ with the deletion of the N-terminal α-helix. However, the enzyme activity at the optimal temperature does not change. It suggests that this N-terminal α-helix is essential for thermostability. Here, the crystal structure of apAPH-△(1-21) has been determined by molecular replacement to 2.5A. A comparison between the two structures suggests a difference in thermostability, and it can be concluded that by adding or deleting a linking structure (located over different domains), the stability or even the activity of an enzyme can be modified.

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

    Directory of Open Access Journals (Sweden)

    Hyun Ho Park

    2009-06-01

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

  17. Purification, crystallization and preliminary x-ray crystallographic studies of RAIDD Death-Domain (DD).

    Science.gov (United States)

    Jang, Tae-ho; Park, Hyun Ho

    2009-06-03

    Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 A and gamma = 120 degrees . The crystals were obtained at room temperature and diffracted to 2.0 A resolution.

  18. Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of Haemophilus influenzae BamD and BamCD complex

    Science.gov (United States)

    Lei, Jintang; Cai, Xun; Ma, Xiaodan; Zhang, Li; Li, Yuwen; Dong, Xue; St Geme, Joseph; Meng, Guoyu

    2015-01-01

    The Bam machinery, which is highly conserved from bacteria to humans, is well recognized as the apparatus responsible for the insertion and folding of most outer membrane proteins in Gram-negative bacteria. In Escherichia coli, the Bam machinery consists of five components (i.e. BamA, BamB, BamC, BamD and BamE). In comparison, there are only four partners in Haemophilus influenzae: a BamB homologue is not found in its genome. In this study, the recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of H. influenzae BamD and BamCD complex are reported. The genes encoding BamC and BamD were cloned into a pET vector and expressed in E. coli. Affinity, ion-exchange and gel-filtration chromatography were used to obtain high-purity protein for further crystallo­graphic characterization. Using the hanging-drop vapour-diffusion technique, BamD and BamCD protein crystals of suitable size were obtained using protein concentrations of 70 and 50 mg ml−1, respectively. Preliminary X-ray diffraction analysis showed that the BamD crystals diffracted to 4.0 Å resolution and belonged to space group P212121, with unit-cell parameters a = 54.5, b = 130.5, c = 154.7 Å. The BamCD crystals diffracted to 3.8 Å resolution and belonged to space group I212121, with unit-cell parameters a = 101.6, b = 114.1, c = 234.9 Å. PMID:25664802

  19. Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

    Indian Academy of Sciences (India)

    Hari S. Misra; Nivedita P. Khairnar; Manjula Mathur; N. Vijayalakshmi; Remesh S. Hire; T. K. Dongre; S. K. Mahajan

    2002-04-01

    A sporulating culture of Bacillus thuringiensis subsp. kenyae strain HD549 is toxic to larvae of lepidopteran insect species such as Spodoptera litura, Helicoverpa armigera and Phthorimaea operculella, and a dipteran insect, Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed in E. coli. The recombinant protein produced 92% mortality in first-instar larvae of Spodoptera litura and 86% inhibition of adult emergence in Phthorimaea operculella, but showed very low toxicity against Helicoverpa armigera, and lower mortality against third-instar larvae of dipteran insects Culex fatigans, Anopheles stephensi and Aedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus thuringiensis var. kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.

  20. 熔融结晶法提纯1-萘酚%Melt Crystallization Purification of 1 -naphthol

    Institute of Scientific and Technical Information of China (English)

    贺小兰; 朱进; 付达权

    2012-01-01

    从混合萘酚中分离提纯1-萘酚采用熔融结晶技术,通过对降温速率、结晶终温、升温速率及“发汗”终温等因素对粗晶体收率及纯度的影响的综合考虑,经多次反复实验,确定了结晶提纯1-萘酚较佳的工艺参数条件,经过单级结晶1-萘酚的含量由80%上升到了93%。%From mixed naphthol in separation and purification of 1 - naphthol by melt crystallization technology, through the cooling rate, the final crystallization temperature, heating rate and the "sweating" the final temperature and other factors on the yield and purity of the crude crystal, the better conditions of process parameters of crystallization purification of 1 - naphthol was determined after repeated experiments, and 1 - napht, hol content was increased from 80% to 93% through a single -stage crystallization.

  1. Cloning, expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody.

    Science.gov (United States)

    Rezaee, Mehdi; Honari, Hossein; Kooshk, Mohammad Reza Ashrafi

    2014-01-01

    Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni(+)-NTA affinity chromatography (95-98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax.

  2. Cloning, expression, crystallization and preliminary structural studies of dihydrodipicolinate reductase from Acinetobacter baumannii

    Science.gov (United States)

    Kaushik, Sanket; Singh, Avinash; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

    2013-01-01

    Acinetobacter baumannii is a virulent pathogenic bacterium that is resistant to most currently available antibiotics. Therefore, the design of drugs for the treatment of infections caused by A. baumannii is urgently required. Dihydrodipicolinate reductase (DHDPR) is an important enzyme which is involved in the biosynthetic pathway that leads to the production of l-lysine in bacteria. In order to design potent inhibitors against this enzyme, its detailed three-dimensional structure is required. DHDPR from A. baumannii (AbDHDPR) has been cloned, expressed, purified and crystallized. Here, the preliminary X-ray crystallographic data of AbDHDPR are reported. The crystals were grown using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitating agent The crystals belonged to the orthorhombic space group P222, with unit-cell parameters a = 80.0, b = 100.8, c = 147.6 Å, and contained four molecules in the asymmetric unit. The complete structure determination of AbDHDPR is in progress. PMID:23722845

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Liaw, M; D' Aragona, F S

    1980-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jaeyong; Feldman, Anat R. [Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, British Columbia, V5A 1S6 (Canada); Delmas, Bernard [Unité de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, F-78350 Jouy-en-Josas (France); Paetzel, Mark, E-mail: mpaetzel@sfu.ca [Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, British Columbia, V5A 1S6 (Canada)

    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.

  6. Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1.

    Science.gov (United States)

    Stocker, Achim; Keis, Stefanie; Cook, Gregory M; Dimroth, Peter

    2005-11-01

    Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.

  7. Gene cloning, expression and purification of human mito-chondrial tRNALeu(UUR) and its mutant

    Institute of Scientific and Technical Information of China (English)

    HAN; Weiguo

    2001-01-01

    [1]Anderson, S., Bankier, A. T., Barrell, B. G. et al., Sequence and organization of the human mitochondrial gene, Nature, 1981, 290(9): 457.[2]Wang, X. M., Yang, Y. S., The mutation of human mitochondrial tRNA and diseases, Chemistry of Life, 1999, 19(4): 171.[3]Goto, Y., Nonaka, I., Horai, S., A mutation in the tRNAleu(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature, 1990, 348(13): 651.[4]Schapira, A. H. V., Mitochondrial disorders, Current Opinion in Genetics and Development, 1993, 3: 457.[5]Moraes, C. T., Ricci, E., Bonilla, W. et al., The mitochondrial tRNAleu(UUR) mutation in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS): genetic, biochemical and morphological correlations in skeletal muscle, Am. J. Hum. Genet., 1992, 50: 934.[6]Chen, L., Jin, Y. X., Wang, D. B. et al., Species-specific identity elements of tRNATrp, Prog. Natl. Sci., 2000, 10(3): 192.[7]Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, 556-558.[8]Merri, C. R., Goldman, D., Keuren, M. L. V., Gel protein stains: silver stain, Methods Enzymol, 1984, 104: 441.[9]Li, Y., Wang, E., Wang, Y., Overproduction and purification of Escherichia coli tRNAleu, Science in China, Ser. C, 1998, 41(3): 225.[10]Hess, J. F., Parisi, M. A., Bennett Clayton, D. A., Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature, 1991, 352: 236.[11]Yousufzai, A. Y. K., Bradford, M. W., Shrago, E. et al., Characterization of the adenine nucleotide translocase of pancreatic islet mitochondria, FEBS Lett., 1982, 137: 201.[12]Welsh, N., Paabo, S., Welsh, M., Decreased mitochondrial gene expression in isolated islet of rats injected neonatally with streptozotocin, Diabetologia, 1991, 34: 626.[13] Chen, L., Jin

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

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Jinlan; Li, Xiaolu [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Feng, Yue; Zhang, Bo [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Miao, Shiying [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Linfang, E-mail: lfwangz@yahoo.com [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Na, E-mail: nawang@tsinghua.edu.cn [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China)

    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 .

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

    Energy Technology Data Exchange (ETDEWEB)

    Jagadeesan, G. [Presidency College, Chennai 600 005 (India); Malathy, P.; Gunasekaran, K. [University of Madras, Chennai 600 025 (India); Harikrishna Etti, S. [GKM College of Engineering and Technology, Kamaraj Salai, Chennai 600 063 (India); Aravindhan, S., E-mail: aravindhanpresidency@gmail.com [Presidency College, Chennai 600 005 (India)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-06-01

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

  12. [Studies on expression, purification, crystal growth and optimization of putative transcription factor LytR from Streptococcus pneumoniae].

    Science.gov (United States)

    Min, Xun; Zhong, Wen; Zhao, Shasha; Dong, Jie; Dong, Shanshan; Zhou, Aie; Yan, Wenjuan; Wang, Deqiang

    2013-08-01

    The aim of the present study was to obtain the crystal of transcription factor LytR of streptococcus pneumoniae for X-ray crystal structure and function analysis. The LytR gene of D39 strains from Streptococcus pneumoniae (S. pn) was cloned into the prokaryotic expression vector pET32a(+), then overexpression was obtained in the E. coli BL21 (DE3) through transformation of the recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with His-tag highly expressed by the induction of 0.5 mmol/L IPTG and was purified by a three step procedure, the purity of the purified LytR recombinant protein was over 90%. Preliminary screening of crystallization conditions was performed using the hanging-drop vapour-diffusing method with Hampton Crystal screen and PEG screen kits. The protein crystals X-ray diffraction data were collected from a single crystal and more stick crystals whose X-ray diffraction reached 4.0 A were obtained. These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.

  13. MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E. Coli AND PURIFICATION

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT pro-tein for futher study. Methods. The gene for encoding hTRT catalytic domain was cloned based on RT-PCR amplification from HeLa cells and sequenced. The cloned hTRTcDNA was in-frame inserted into His-tag fusion expression vector pEK318. The His-tag hTRT fusion proteins were purified by Ni-NTA chromatography and stained by westerm blotting. Results. An approximately 620bp fragment was generated and cloned into pBluescript SK + between Sail and BamHI sites. DNA sequencing showed the isolated fragment was consistem to those reported. SDS-PAGE present that a 17kDa protein was expressed stably in E. coli JM109 harboring pEKTRTM4 containing 6 × His-tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6 × His-tag and hTRT 243aa was only detectable as 27 kDa band in western blotting. Both of fu-sion proteins were purified by Ni-NTA chromatography and showed single band( > 95% purifity) in Coomassie Bril-liant staining. Westem-blotting confirmed that two proteins could be recognized by the Ni-NTA AP conjugate. Conclusions. The hTRT catalytic domain was highly conserved. The expressed hTRT protein contained recogniz-able His-tag, telomerase-specific and strong antigenic epitops, which may be convenient for further investigation.

  14. Cloning, large-scale production, and purification of active dimeric rat vascular endothelial growth factor (rrVEGF-164).

    NARCIS (Netherlands)

    Geutjes, P.J.; Nillesen, S.T.M.; Lammers, G.; Daamen, W.F.; Kuppevelt, A.H.M.S.M. van

    2010-01-01

    Large-scale production of recombinant rat vascular endothelial growth factor (rrVEGF-164) is desirable for angiogenic studies. In this study, biologically active recombinant rat vascular endothelial growth factor (rrVEGF-164) was cloned and expressed in the yeast Pichia pastoris, and large-scale pro

  15. Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39.

    Science.gov (United States)

    Zhong, Xiaotian; Buddha, Madhavan; Guidotti, Guido; Kriz, Ron; Somers, Will; Mosyak, Lidia

    2008-11-01

    CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction-quality crystals of sCD39 were produced by the vapor-diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P3(2), with unit-cell parameters a = b = 118.1, c = 81.6 A and with two sCD39 copies in the asymmetric unit. Several low- to medium-resolution diffraction data sets were collected using an in-house X-ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular-replacement search was performed against the complete 3.2 A data set using a maximum-likelihood molecular-replacement method as implemented in Phaser. The initial model of the two sCD39 monomers was placed into the P3(2) lattice and rigid-body refined and position-minimized with PHENIX.

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

    Energy Technology Data Exchange (ETDEWEB)

    Renzi, Fabiana; Panetta, Gianna; Vallone, Beatrice; Brunori, Maurizio [Dipartimento di Scienze Biochimiche, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy); Arceci, Massimo [Istituto di Biologia e Patologia Molecolari CNR, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy); Bozzoni, Irene [Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy); Istituto di Biologia e Patologia Molecolari CNR, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy); Dipartimento di Genetica e Biologia Molecolare, University of Rome ‘La Sapienza’, Piazalle Aldo Moro 5, 00185 Roma (Italy); Laneve, Pietro [Istituto di Biologia e Patologia Molecolari CNR, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy); Dipartimento di Genetica e Biologia Molecolare, University of Rome ‘La Sapienza’, Piazalle Aldo Moro 5, 00185 Roma (Italy); Caffarelli, Elisa, E-mail: elisa.caffarelli@uniroma1.it [Istituto di Biologia e Patologia Molecolari CNR, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy); Dipartimento di Scienze Biochimiche, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy)

    2006-03-01

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

  17. Purification and crystallization of the yeast translation elongation factor eEF3

    DEFF Research Database (Denmark)

    Andersen, Christian Folsted; Anand, Monika; Boesen, Thomas

    2004-01-01

    A Saccharomyces cerevisiae strain expressing full-length histidine-tagged translation elongation factor 3 (eEF3) as the only form of the protein facilitated purification of the factor for both structural and functional studies. Additionally, an identical full-length form has been successfully...

  18. Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E.coil

    Institute of Scientific and Technical Information of China (English)

    WANG Rui-jian; YANG Xue-ying; ZHENG Liang-yu; YANG Ye; GAO Gui; CAO Shu-gui

    2008-01-01

    The entire gene of carboxyltransferase(CT) domain of acetyl-CoA carboxylase(ACCase) from Chinese Spring wheat(CSW) plastid was cloned firstly,and the 2.3 kb gene was inserted into PET28a+ vector and expressed in E.coil in a soluble state.The (His)6 fusion protein was identified by SDS-PAGE and Western blot.The recombinant protein was purified by affinity chromatography,and the calculated molecular mass(Mr) was 88000.The results of the sequence analysis indicate that the cloned gene(GeneBank accession No.EU124675) was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid.The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides,and further to screen new herbicides.

  19. Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host

    OpenAIRE

    2014-01-01

    Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak a...

  20. 4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene.

    Science.gov (United States)

    Czempinski, K; Kruft, V; Wöstemeyer, J; Burmester, A

    1996-09-01

    We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bagautdinov, Bagautdin; Kunishima, Naoki, E-mail: kunisima@spring8.or.jp [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

    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.

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

    Science.gov (United States)

    Rodamilans, Bernardo; Montoya, Guillermo

    2007-04-01

    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(1), with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 A, alpha = gamma = 90, beta = 101.02 degrees , and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 A using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).

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

    Science.gov (United States)

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

    2012-12-01

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

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

    Science.gov (United States)

    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.

  5. Expression,purification,crystallization,and preliminary X-ray diffraction analysis of the human TLE1 Q domain

    Institute of Scientific and Technical Information of China (English)

    Su Wang; Jiamu Du; Hua Tang; Xinyu Ding; Manwu Zha; Zhifei Xu

    2011-01-01

    Human transducin-like enhancer of split 1(TLE1)plays crucial roles in a number of developmental processes and is involved in pathogenesis of malignancy tumors.The N-terminal glutamine-rich domain(Q domain)of TLEI mediates its tetramerization and interactions with different DNA-binding transcription factors to regulate Notch and Wnt signaling pathways.To better understand the molecular mechanism of TLEI's functions in these pathways,we cloned,purified,and crystallized the TLE1 Q domain(TLEI-Q).The crystals belong to space group C2221,with the complete diffraction data of the native and Se-Met TLEI-Q collected to 3.5 and 4.1 A resolutions,respectively.The phasing-solving and model building are in progress.

  6. A carboxymethyl cellulase from a marine yeast ( Aureobasidium pullulans 98): Its purification, characterization, gene cloning and carboxymethyl cellulose digestion

    Science.gov (United States)

    Rong, Yanjun; Zhang, Liang; Chi, Zhenming; Wang, Xianghong

    2015-10-01

    We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase (CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U (mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40°C, much lower than that of the CMCases from other fungi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity (pH 5.0-6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. K m and V max values of the purified enzyme were 4.7 mg mL-1 and 0.57 µmol L-1 min-1 (mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose (CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp (EU978473). The protein deduced contained the conserved domain of cellulase superfamily (glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

  7. Overexpression, purification, crystallization and data collection of Sulfolobus solfataricus Sso6206, a novel highly conserved protein

    Energy Technology Data Exchange (ETDEWEB)

    McEwan, Andrew R.; Liu, Huanting; Oke, Muse; Carter, Lester; Powers, Helen; Dorward, Mark; McMahon, Stephen A.; White, Malcolm F.; Naismith, James H., E-mail: naismith@st-and.ac.uk [The Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST,Scotland (United Kingdom)

    2006-03-01

    The novel protein Sso6206 has been crystallized; interestingly, the protein may form large multi-subunit oligomers. Sso6206, a 10.5 kDa protein from Sulfolobus solfataricus, has been overexpressed, purified and crystallized. The protein crystallizes in space group P6{sub 1/5}22, with unit-cell parameters a = b = 157.8, c = 307.3 Å. The crystals are hexagonal bipyramids and a data set has been collected to 2.4 Å resolution. Molecular replacement cannot be attempted as no convincing model can be identified. Crystals of selenomethionine-variant protein have not yet been obtained. Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

  8. Purification, characterization and molecular cloning of Cha o 1, a major allergen of Chamaecyparis obtusa (Japanese cypress) pollen.

    Science.gov (United States)

    Suzuki, M; Komiyama, N; Itoh, M; Itoh, H; Sone, T; Kino, K; Takagi, I; Ohta, N

    1996-01-01

    Pollen of Chamaecyparis obtusa (Japanese cypress) is one of the causes of allergic pollinosis in Japan. A major allergen of the pollen designated Cha o 1, was purified by two-step ion exchange chromatography. Cha o 1 was separated into four components with molecular masses of 48.5 kDa and 52.0 kDa, each with pIs of 6.77 and 6.82. The 23-residue N-terminal sequence of Cha o 1 was determined and shown to have high identity with that of Cry j 1, a major allergen of Cryptomeria japonica pollen. cDNA coding for Cha o 1 was cloned by hybridization screening using Cry j 1 cDNA as a probe. One of the cDNA clones, pCHA-1 was sequenced and found to code for a putative 21-residue signal peptide and a 354-residue native protein with a derived molecular mass of 38.1 kDa. The deduced amino acid sequence of Cha o 1 showed 79-80% identity with those of Cry j 1. These findings were consistent with observations of a close crossreaction between the two allergens. Homology analyses revealed that Cha o 1 had 46-49% identity with Amb a 1 families and Amb a 2, the major allergens of short ragweed. Cry j 1 has pectate lyase enzyme activity, suggesting that Cha o 1 may have the same enzyme activity as Cry j 1.

  9. Cloning, purification and preliminary crystallographic analysis of a putative DNA-binding membrane protein, YmfM, from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ling; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rice, David W., E-mail: d.rice@sheffield.ac.uk [Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN (United Kingdom)

    2008-07-01

    Truncation by the removal of the C-terminal hydrophobic transmembrane anchor has enabled the overexpression of a soluble domain of S. aureus YmfM in Escherichia coli, which has then been purified and subsequently crystallized. The Staphylococcus aureus protein YmfM contains a helix–turn–helix motif and is thought to be a putative DNA-binding protein which is associated with the membrane through a C-terminal hydrophobic transmembrane anchor. Truncation of the protein by the removal of this C-terminal hydrophobic segment has enabled the overexpression of a soluble domain of S. aureus YmfM (ΔYmfM) in Escherichia coli, which has been purified and subsequently crystallized. Crystals of ΔYmfM diffract to beyond 1.0 Å resolution and belong to one of the pair of enantiomorphic tetragonal space groups P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 45.5, c = 72.9 Å and one molecule in the asymmetric unit. The crystals of ΔYmfM have an unusually low V{sub M} of 1.6 Å{sup 3} Da{sup −1}, which is one of the lowest values observed for any protein to date. A full structure determination is under way in order to provide insights into the function of this protein.

  10. Purification, characterization and cDNA cloning of an analgesic peptide from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU2).

    Science.gov (United States)

    Zhang, R; Yang, Z; Liu, Y F; Cui, Y; Zhang, J H

    2011-01-01

    In this study an analgesic peptide was purified through five continuous chromatographic steps. The mouse twisting model test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU2 was further qualified by Reverse Phase-High Performance Liquid Chromatography and High Performance Capillary Electrophoresis. The molecular weight, isoelectric point, and N-terminal sequence of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was cloned by rapid amplification of the cDNA ends from the cDNA pool of scorpion glands. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK alpha2 (GenBank Acc. No. AF288608) and BmK alphaTX11 (GenBank Acc. No. AF155364). We report herein a purification procedure that yields substantial amounts of natural BmK AGP-SYPU2 with high analgesic activity.

  11. Biodiversity of multiple Pregnancy-Associated Glycoprotein (PAG) family: gene cloning and chorionic protein purification in domestic and wild eutherians (Placentalia)--a review.

    Science.gov (United States)

    Szafranska, Bozena; Panasiewicz, Grzegorz; Majewska, Marta

    2006-01-01

    This review presents a broad overview of chorionic glycoproteins encoded by the Pregnancy-Associated Glycoprotein (PAG) gene family and also serves to illustrate how the recent discovery of the PAG family has contributed to our general knowledge of genome evolution, placental transcription and placental protein expression. The complex and large PAG family is restricted to the Artiodactyla order, although single PAG-like genes have also been identified in species outside the Artiodactyla. The PAGs are members of the aspartic proteinase (AP) superfamily. Unexpectedly, however, some members of the PAG family possess amino acid substitutions within and around the active site that likely render them unable to act as proteinases. This paper summarises the available information regarding biodiversity of PAG gene expression based on cDNA cloning, mRNA localisation studies and the structural organisation of the PAG genes with a particular emphasis on PAG promoters. It also compares available data regarding PAG protein purifications, sequencing and their N-glycodiversity. Finally, it discusses the scientific relevance, possible functional roles of the PAGs and describes possible profitable applications related to the detection of PAG proteins in the blood of pregnant domestic and wild species.

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

    Science.gov (United States)

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

    2005-02-01

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

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

    Science.gov (United States)

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

    2005-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Gohain, Neelakshi [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany); Thomashow, Linda S. [USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, Pullman, Washington 99164-6430 (United States); Mavrodi, Dmitri V. [Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430 (United States); Blankenfeldt, Wulf, E-mail: wulf.blankenfeldt@mpi-dortmund.mpg.de [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)

    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.

  15. Purification, molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon.

    Science.gov (United States)

    Nagatomo, Yoshihisa; Usui, Shiori; Ito, Takamitsu; Kato, Akira; Shimosaka, Makoto; Taguchi, Goro

    2014-11-01

    C-Glycosides are characterized by their C-C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C-Glycosides are remarkably stable, as their C-C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C-glycosylflavonoids; however, the genes encoding for enzymes that catalyze C-glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C-glucosyltransferase gene from the dicot plant Fagopyrum esculentum M. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C-glucosylation of 2-hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C-glucosylation activity towards 2-hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2',4',6'-trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C-glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases.

  16. Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker.

    Science.gov (United States)

    Kawashima, Satoshi; Ikehata, Hiroki; Tada, Chihiro; Ogino, Tomohiro; Kakizaki, Hiromi; Ikeda, Mana; Fukushima, Hideto; Matsumiya, Masahiro

    2016-01-20

    Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)₈-fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

  17. Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus).

    Science.gov (United States)

    Zhou, Long-Zhen; Ruan, Mi-Mi; Cai, Qiu-Feng; Liu, Guang-Ming; Sun, Le-Chang; Su, Wen-Jin; Cao, Min-Jie

    2012-03-01

    A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40°C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45°C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M(-1). Apparent K(m) was 1.02 μM and k(cat) was 148 S(-1) for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5'-RACE and 3'-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.

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

    Science.gov (United States)

    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°.

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

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Tengchuan; Howard, Andrew; Zhang, Yu-Zhu, E-mail: zhangy@iit.edu [Department of Biology, Illinois Institute of Technology, Chicago, IL 60616 (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sanctis, Daniele de; Rêgo, Ana T.; Marçal, David; McVey, Colin E.; Carrondo, Maria A. [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal); Enguita, Francisco J., E-mail: fenguita@fm.ul.pt [Instituto de Medicina Molecular, Avenida Professor Egas Moniz, 1649-028 Lisboa (Portugal); Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bajaj, Mamta [School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States); Moriyama, Hideaki, E-mail: hmoriyama2@unl.edu [Department of Chemistry, e-Toxicology and Biotechnology, University of Nebraska-Lincoln, Hamilton Hall, Lincoln, Nebraska 68588-0304 (United States); School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States)

    2007-05-01

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

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Abramchik, Yu. A., E-mail: inna@ns.crys.ras.ru; Timofeev, V. I., E-mail: espiov@ibch.ru; Zhukhlistova, N. E., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    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.

  4. Purification, crystallization and preliminary X-ray diffraction analysis of a plant subtilase.

    Science.gov (United States)

    Rose, Rolf; Huttenlocher, Franziska; Cedzich, Anna; Kaiser, Markus; Schaller, Andreas; Ottmann, Christian

    2009-05-01

    The subtilase SBT3 from Solanum lycopersicum (tomato) was purified from a tomato cell culture and crystallized using the sitting-drop vapour-diffusion method. A native data set was collected to 2.5 A resolution at 100 K using synchrotron radiation. For experimental phasing, CsCl-derivative and tetrakis(acetoxymercuri)methane (TAMM) derivative crystals were employed for MIRAS phasing. Three caesium sites and one TAMM site were identified, which allowed solution of the structure.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Minako; Suzuki, Nobuo N.; Fujioka, Yuko [Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-12, W-6, Kita-ku, Sapporo 060-0812 (Japan); Ohsumi, Yoshinori [Department of Cell Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki 444-8585 (Japan); Inagaki, Fuyuhiko, E-mail: finagaki@pharm.hokudai.ac.jp [Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-12, W-6, Kita-ku, Sapporo 060-0812 (Japan)

    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. Purification, amino acid sequence, and cDNA cloning of trypsin inhibitors from onion (Allium cepa L.) bulbs.

    Science.gov (United States)

    Deshimaru, Masanobu; Watanabe, Akira; Suematsu, Keiko; Hatano, Maki; Terada, Shigeyuki

    2003-08-01

    Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3 x 10(-9) M, 2.3 x 10(-7) M, and 3.1 x 10(-7) M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.

  8. Purification, cDNA cloning, and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense).

    Science.gov (United States)

    Inamine, Saki; Onaga, Shoko; Ohnuma, Takayuki; Fukamizo, Tamo; Taira, Toki

    2015-01-01

    Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.

  9. Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster.

    Science.gov (United States)

    Nagasawa, Tatsuki; Sano, Kaori; Kawaguchi, Mari; Kobayashi, Ken-Ichiro; Yasumasu, Shigeki; Inokuchi, Tomofumi

    2016-04-01

    Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.

  10. Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).

    Science.gov (United States)

    Pei, Zhihua; Bian, Lu; Zhang, Hui; Gao, Yunhang; Ma, Hongxia

    2014-01-01

    Musca domestica L. (Diptera: Muscidae), the housefly, exhibits unique immune defenses and can produce antibacterial substances upon stimulation with bacteria. On the basis of the cDNA library constructed using the suppression subtractive hybridization method, a 1188-bp antibacterial substance gene, which we named AS566, was amplified by rapid amplification of cDNA ends from M. domestica larva stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In this study, the full-length AS566 gene was cloned and inserted into a His-tagged Escherichia coli (Enterobacteriaceae: Escherichia) prokaryotic expression system to enable production of the recombinant protein. The recombinant AS566 protein was purified in denatured form from inclusion bodies and renatured to obtain functionally active AS566 protein. The bacteriostatic activity of the recombinant purified AS566 protein was assessed using the Oxford plate assay system and the results indicated that AS566 had antibacterial activity against six bacteria, including an E. coli clinical isolate, S. pullorum, Streptococcus bovis (Streptococcaceae: Streptococcus), Streptococcus suis, and Staphylococcus aureus (Staphylococcaceae: Staphylococcus) in vitro. The antibacterial activity of AS566 toward Gram- bacteria was two times greater than that against Gram+ bacteria. The sequencing results and BLAST analysis showed that the antibacterial substance gene AS566 was not homologous to any other antibacterial substance genes in GenBank. The antibacterial mechanisms of the newly discovered AS566 protein warrant further study.

  11. Gene cloning, purification, and characterization of two cyanobacterial NifS homologs driving iron-sulfur cluster formation.

    Science.gov (United States)

    Kato, S; Mihara, H; Kurihara, T; Yoshimura, T; Esaki, N

    2000-11-01

    Iron-sulfur proteins are essential in the photosynthetic system and many other biological processes. We have isolated and characterized enzymes driving the formation of iron-sulfur clusters from Synechocystis sp. PCC6803. Two genes (slr0387 and sll0704), showing similarity to nifS of Azotobacter vinelandii, were cloned, and their gene products (SsCsdl and SsCsd2) were purified. They catalyzed the desulfuration of L-cysteine. Reconstitution of a [2Fe-2S] cluster of cyanobacterial ferredoxin proceeded much faster in the presence of L-cysteine and either of these enzymes than when using sodium sulfide. These results suggest that SsCsdl and SsCsd2 facilitate the iron-sulfur cluster assembly by producing inorganic sulfur from L-cysteine. Synechocystis sp. PCC6803 has no gene coding for a protein with similarity to the N-terminal domain of NifU of A. vinelandii, which is believed to cooperate with NifS to assemble iron-sulfur clusters. Thus, the cluster formation in the cyanobacterium probably proceeds through a mechanism that is different from that in A. vinelandii.

  12. Purification and cDNA cloning of a novel neurotoxic peptide (Acra3) from the scorpion Androctonus crassicauda.

    Science.gov (United States)

    Caliskan, Figen; García, Blanca I; Coronas, Fredy I V; Restano-Cassulini, Rita; Korkmaz, Ferhan; Sahin, Yalcin; Corzo, Gerardo; Possani, Lourival D

    2012-09-01

    Androctonus crassicauda is one of the Southeastern Anatolian scorpions of Turkey with ethno-medical and toxicological importance. Two toxic peptides (Acra1 and Acra2) were isolated and characterized from the venom of this scorpion. In this communication, the isolation of an additional toxin (Acra3) by chromatographic separations (HPLC and TSK-gel sulfopropyl) and its chemical and functional characterization is reported. Acra3 is a 7620Da molecular weight peptide, with 66 amino acid residues crosslinked by four disulfide bridges. The gene coding for this peptide was cloned and sequenced. Acra3 is anticipated to undergo post-translational modifications at the C-terminal region, having an amidated serine as last residue. Injection of Acra3 induces severe neurotoxic events in mice, such as: excitability and convulsions, leading to the death of the animals within a few minutes after injection. Electrophysiological assays conducted with pure Acra3, using cells that specifically expressed sodium channels (Nav1.1-Nav1.6) showed no clear effect. The exact molecular target of Acra3 remained undiscovered, similar to three other scorpion peptides that clustered very closely in the phylogenetic tree included here. The exact target of these four peptides is not very clear.

  13. The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

    Science.gov (United States)

    Brochu, Denis; Vadeboncoeur, Christian

    1999-01-01

    In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg2+ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its Kms for HPr and ATP are 31 μM and 1 mM, respectively. PMID:9922231

  14. Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria.

    Science.gov (United States)

    Campos, Eleonora; Negro Alvarez, María José; Sabarís di Lorenzo, Gonzalo; Gonzalez, Sergio; Rorig, Marcela; Talia, Paola; Grasso, Daniel H; Sáez, Felicia; Manzanares Secades, Paloma; Ballesteros Perdices, Mercedes; Cataldi, Angel A

    2014-01-01

    The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40 °C and Km and Kcat values were 2.92 mM and 1.32 seg(-1), respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s(-1). These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. (Pfizer)

    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.

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

    Science.gov (United States)

    Bagautdinov, Bagautdin; Kunishima, Naoki

    2006-04-01

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

  17. Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb (Amaryllidaceae)

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A novel mannose-binding agglutinin was purified from bulbs of Zephyranthes candida Herb by extraction,precipitation with 80% (NH4)2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel filtration on Sephacryl S-100. The purified Z. candidaagglutinin (ZCA) migrated as a single band of 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the lectin, as determined by gel filtration chromatography, was 48 kDa. The results indicated that ZCA was composed of four identical subunits of 12 kDa each (homotetrameric nature). The ZCA agglutinated rabbit erythrocytes, Escherichia coli and Saccharomyces cerevisiae cells at concentrations of 0.95, 1.90,and 31.30 μg/mL, respectively. Bioassays indicated that ZCA has a significant effect on wheat aphid survival.Mortality after 7 d was > 90% at 0.26%. A degenerate primer was designed in accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3'- and 5'-rapid amplification of cDNA ends.The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa.The result show that the zca gene encodes a protein precursor with a signal peptide, a mature protein, and a Cterminal cleavage amino acids sequence. Molecular modeling of ZCA indicated that its three-dimensional structure strongly resembles that of the snowdrop agglutinin. Blocks' analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes (QDNY).

  18. Purification and Characterization of a Novel Thermostable Chitinase from Thermomyces lanuginosus SY2 and Cloning of Its Encoding Gene

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodeeyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 min. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer (pH 2.5). The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable ehitinases so far isolated in fungi. Ca2+, Ba2+, Na2+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, Hg2+, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplieation. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.

  19. Purification, cloning, expression, and biochemical characterization of a monofunctional catalase, KatP, from Pigmentiphaga sp. DL-8.

    Science.gov (United States)

    Dong, Weiliang; Hou, Ying; Li, Shuhuan; Wang, Fei; Zhou, Jie; Li, Zhoukun; Wang, Yicheng; Huang, Fei; Fu, Lei; Huang, Yan; Cui, Zhongli

    2015-04-01

    Catalases are essential components of the cellular equipment used to cope with oxidative stress. The monofunctional catalase KatP was purified from Pigmentiphaga sp. using ammonium sulfate precipitation (ASP), diethylaminoethyl ion exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC). The purified catalase formed polymer with an estimated monomer molecular mass of 54kDa, which were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. KatP exhibited a specific catalytic activity of 73,000U/mg, which was higher than that of catalase-1 of Comamonas terrigena N3H (55,900U/mg). Seven short tryptic fragments of this catalase were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS/MS), and the gene, katP, was cloned by PCR amplification and overexpressed in Escherichia coli BL21 (DE3). Based on the complete amino acid sequence, KatP was identified as a clade 3 monofunctional catalase. The specific activities of recombinant KatP for hydrogen peroxide (690,000U/mg) increased 9-fold over that of the parent strain. The Km and Vmax of recombinant KatP were 9.48mM and 81.2mol/minmg, respectively. The optimal pH and temperature for KatP were 7.0 and 37°C, respectively, and the enzyme displayed abroad pH-stable range of 4.0-11.0. The enzyme was inhibited by Zn(2+), Cu(2+), Cr(2+), and Mn(2+), whereas Fe(3+) and Mg(2+) stimulated KatP enzymatic activity. Interestingly, the catalase activity of recombinant KatP displayed high stability under different temperature and pH conditions, suggesting that KatP is a potential candidate for the production of catalase.

  20. Purification and characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli.

    Science.gov (United States)

    Liebl, W; Feil, R; Gabelsberger, J; Kellermann, J; Schleifer, K H

    1992-07-01

    Maltodextrin glycosyltransferase (4-alpha-glucanotransferase) of the extremely thermophilic ancestral bacterium Thermotoga maritima has been purified from an Escherichia coli clone expressing the corresponding T. maritima MSB8 chromosomal gene. T. maritima 4-alpha-glucanotransferase, an approximately 53-kDa monomeric enzyme, is the most thermophilic glycosyltransferase described to date. It retained more than 90% of its maximum activity at temperatures from 55 degrees C up to 80 degrees C. The proposed action modus is the transfer of 1,4-alpha-glucanosyl chains, thus resulting in the disproportionation of 1,4-alpha-glucans. It converted soluble starch, amylopectin, and amylose, thereby changing the iodine staining properties of these substrates. The addition of low-molecular-mass malto-oligosaccharides, which act as glucanosyl acceptor molecules, enhanced the reaction and resulted in the formation of a series of linear maltohomologues from two to more than nine glucose units in size. Use of either of the malto-oligosaccharides maltotetraose, maltopentaose, maltohexaose, or maltoheptaose as sole substrate also yielded linear maltohomologues. On the other hand, maltose and maltotriose were not disproportionated by 4-alpha-glucanotransferase, although both were good acceptors for glucanosyl transfer. Glucose did not function as an acceptor in transfer reactions. Glucose also never appeared as a reaction product. The chain length of glucanosyl segments transferred ranged from two to probably far more than six glucose residues. Comparison of the N-terminal amino acid sequence of 4-alpha-glucanotransferase with other published protein sequences revealed significant similarity to sequences near the N-termini of various eucaryotic maltases and bacterial cyclodextrin glycosyltransferases, suggesting its relatedness on the molecular level with other starch- and maltodextrin-converting enzymes.

  1. Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi

    Directory of Open Access Journals (Sweden)

    Karan Ram

    2013-01-01

    Full Text Available Abstract Background Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures. Results Analysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 β-galactosidase gene, named bga. In order to study the biochemical properties of the β-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2 gene promoter. The recombinant β-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (−5 to 60°C with an optimum of 50°C, and 10% of its maximum activity at 4°C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic β-galactosidase was also stable and active in 10–20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol. Conclusion The H. lacusprofundi β-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is

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

    Energy Technology Data Exchange (ETDEWEB)

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro; Shimizu, Hironari [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Nara, Takeshi; Aoki, Takashi [Department of Parasitology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Harada, Shigeharu [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Kita, Kiyoshi, E-mail: kitak@m.u-tokyo.ac.jp [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    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%.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bacik, John-Paul; Brigley, Angela M.; Channon, Lisa D.; Audette, Gerald F.; Hazes, Bart, E-mail: bart.hazes@ualberta.ca [Department of Medical Microbiology and Immunology, 1-15 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7 (Canada)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Srimathi, Thiagarajan; Robbins, Sheila L.; Dubas, Rachel L. [Basic Science, Fox Chase Cancer Center, Philadelphia, PA 19111 (United States); Seo, Jang-Hoon [Department of Clinical Laboratory Science, Shinheung College, Uijeongbu, Kyungki-Do 480-701 (Korea, Republic of); Park, Young Chul, E-mail: young.park@fccc.edu [Basic Science, Fox Chase Cancer Center, Philadelphia, PA 19111 (United States)

    2007-01-01

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

  7. Purification of 2,6-Diisopropylnaphthalene by Static Melt Crystallization from a Mixture Containing Diisopropylnaphthalene Isomers

    Institute of Scientific and Technical Information of China (English)

    TIAN,Zheng-Hua; JIA,Hong-Min; ZHAO,Gui-Li; HU,Wei-Qi

    2007-01-01

    2,6-Diisopropylnaphthalene (2,6-DIPN), as the precursor of important monomer 2,6-naphthalene dicarboxylic acid, could be produced by the shape-selective isopropylation of naphthalene with propene resulting in an isomeric mixture having different alkylation levels. Since the boiling points of DIPNs were very close and the differences of melting points in-between isomers were quite distinctive, the static melt crystallization was applied to separate and purify 2,6-DIPN from its isomers. 2,6-DIPN with purity ≥99% was produced through a process of three stages:crystallization→sweating→melting. The phase diagram of 2,6-DIPN-2,7-DIPN binary system was plotted to optimize the temperature control of crystallization. By repeated crystallization of melts with different concentration levels, the yield of pure 2,6-DIPN could be enhanced to 87%. No solvent was necessary.

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

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Tengchuan; Chen, Yu-Wei; Howard, Andrew; Zhang, Yu-Zhu, E-mail: zhangy@iit.edu [Department of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616 (United States)

    2008-07-01

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

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

    Science.gov (United States)

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

    2013-04-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Misra, Gauri [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Aggarwal, Anita [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Mittal, Sonia [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Singh, Yogendra [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Ramachandran, Ravishankar, E-mail: r-ravishankar@cdri.res.in [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India)

    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%.

  11. Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core.

    Science.gov (United States)

    Tomiya, Noboru; Narang, Someet; Park, Jung; Abdul-Rahman, Badarulhisam; Choi, One; Singh, Sundeep; Hiratake, Jun; Sakata, Kanzo; Betenbaugh, Michael J; Palter, Karen B; Lee, Yuan C

    2006-07-14

    Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

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

    Science.gov (United States)

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

    2009-04-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-02

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-07-20

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Neil G., E-mail: neison@chem.gla.ac.uk; Riboldi-Tunicliffe, Alan [Department of Chemistry and WestCHEM, Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom); Mitchell, Timothy J. [Division of Infection and Immunity (IBLS), Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom); Isaacs, Neil W. [Department of Chemistry and WestCHEM, Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

    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.

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

    Science.gov (United States)

    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).

  17. Purification and Crystallization of Flammulin, a Basic Protein with Anti-tumor Activities from Flammulina Velutipes

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Flammulin, an anti-tumor protein, was purified from the aqueous extract of basidiomes of Flammulina Velutipes to electrophoretic homogeneity and crystallized by microdialysis against a polyethylene glycol- sodium phosphate buffer. The purified product was found to have marked antitumor effects and be able to affect the tumor cells directly.

  18. Purification, crystallization and preliminary X-ray diffraction analysis of an archaeal ABC-ATPase

    NARCIS (Netherlands)

    Verdon, Grégory; Albers, Sonja-V.; Dijkstra, Bauke W.; Driessen, Arnold J.M.; Thunnissen, Andy-Mark W.H.

    2002-01-01

    In the archaeon Sulfolobus solfataricus glucose uptake is mediated by an ABC transport system. The ABC-ATPase of this transporter (GlcV) has been overproduced in Escherichia coli and purified. Crystals of GlcV suitable for data collection were obtained in the absence of nucleotide by microseeding

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

    Energy Technology Data Exchange (ETDEWEB)

    Kelleher, Alan; Liu, Zhuyun; Seid, Christopher A.; Zhan, Bin; Asojo, Oluwatoyin A., E-mail: asojo@bcm.edu [Baylor College of Medicine, 1102 Bates Avenue, Suite 550, Mail Stop BCM320, Houston, TX 77030-3411 (United States)

    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.

  20. A novel cold-active β-D-galactosidase from the Paracoccus sp. 32d - gene cloning, purification and characterization

    Directory of Open Access Journals (Sweden)

    Wierzbicka-Woś Anna

    2011-12-01

    Full Text Available Abstract Background β-D-Galactosidases (EC 3.2.1.23 catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Cold-active β-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis. Results A genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the β-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-β-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable

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

    Energy Technology Data Exchange (ETDEWEB)

    Rivas, Blanca de las; Rodríguez, Héctor [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Angulo, Iván [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Muñoz, Rosario [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Mancheño, José M., E-mail: xjosemi@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain)

    2007-07-01

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

  2. The quorum-quenching lactonase from Alicyclobacter acidoterrestris : purification, kinetic characterization, crystallization and crystallographic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Bergonzi, Celine; Schwab, Michael; Chabriere, Eric; Elias, Mikael

    2017-07-26

    Lactonases comprise a class of enzymes that hydrolyze lactones, including acyl-homoserine lactones (AHLs); the latter are used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases have therefore been demonstrated to quench AHL-based bacterial communication. In particular, lactonases are capable of inhibiting bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. A novel representative from the metallo-β-lactamase superfamily, named AaL, was isolated from the thermoacidophilic bacteriumAlicyclobacter acidoterrestris. Kinetic characterization proves AaL to be a proficient lactonase, with catalytic efficiencies (kcat/Km) against AHLs in the region of 105M-1s-1. AaL exhibits a very broad substrate specificity. Its structure is expected to reveal the molecular determinants for its substrate binding and specificity, as well as to provide grounds for future protein-engineering projects. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection of AaL at 1.65Å resolution are reported.

  3. Phosphate-binding protein from Polaromonas JS666: purification, characterization, crystallization and sulfur SAD phasing

    Energy Technology Data Exchange (ETDEWEB)

    Pegos, Vanessa R.; Hey, Louis; LaMirande, Jacob; Pfeffer, Rachel; Lipsh, Rosalie; Amitay, Moshe; Gonzalez, Daniel; Elias, Mikael (JCT-Israel); (UMM); (CNRS-UMR)

    2017-05-25

    Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP fromPolaromonasJS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently known PBPs. Here, it is shown that the PBP fromPolaromonasJS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Å resolution of the PBP fromPolaromonasJS666 are reported.

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

    Energy Technology Data Exchange (ETDEWEB)

    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}.

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

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Feng; Jin, Tengchuan; Howard, Andrew; Zhang, Yu-Zhu, E-mail: zhangy@iit.edu [Department of Biology, Illinois Institute of Technology, Chicago, IL 60616 (United States)

    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.

  6. Expression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae pseudopilin EpsH

    Energy Technology Data Exchange (ETDEWEB)

    Raghunathan, Kannan; Vago, Frank S.; Ball, Terry; Yakubova, Nafissa; Grindem, David; Wedemeyer, William J.; Arvidson, Dennis N.; (MSU)

    2010-01-12

    EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.39, b = 71.11, c = 84.64 {angstrom}. There were two protein molecules in the asymmetric unit, which gave a Matthews coefficient V{sub M} of 2.1 {angstrom}{sup 3} Da{sup -1}, corresponding to 41.5% solvent content.

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

    Energy Technology Data Exchange (ETDEWEB)

    Jens, Jason; Raghunathan, Kannan; Vago, Frank; Arvidson, Dennis; (MSU)

    2010-01-12

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

  8. Expression, purification and crystallization of the ancestral androgen receptor-DHT complex.

    Science.gov (United States)

    Colucci, Jennifer K; Ortlund, Eric A

    2013-09-01

    Steroid receptors (SRs) are a closely related family of ligand-dependent nuclear receptors that mediate the transcription of genes critical for development, reproduction and immunity. SR dysregulation has been implicated in cancer, inflammatory diseases and metabolic disorders. SRs bind their cognate hormone ligand with exquisite specificity, offering a unique system to study the evolution of molecular recognition. The SR family evolved from an estrogen-sensitive ancestor and diverged to become sensitive to progestagens, corticoids and, most recently, androgens. To understand the structural mechanisms driving the evolution of androgen responsiveness, the ancestral androgen receptor (ancAR1) was crystallized in complex with 5α-dihydrotestosterone (DHT) and a fragment of the transcriptional mediator/intermediary factor 2 (Tif2). Crystals diffracted to 2.1 Å resolution and the resulting structure will permit a direct comparison with its progestagen-sensitive ancestor, ancestral steroid receptor 2 (AncSR2).

  9. Purification, crystallization and preliminary X-ray crystallographic studies of Drep2 CIDE domain.

    Science.gov (United States)

    Lee, Seung Mi; Park, Hyun Ho

    2014-10-01

    Drep2 is a novel nuclease from the fruit fly that might have a similar function in apoptosis to DFF40 and DFF45, which are primary players in apoptotic DNA fragmentation. Drep2 contains a conserved CIDE domain of ∼90 amino-acid residues that is involved in protein-protein interaction. In this study, the Drep2 CIDE domain was purified and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were then collected to a resolution of 2.3 Å. The crystals were found to belong to the orthorhombic space group P212121, with unit-cell parameters a = 50.28, b = 88.70, c = 113.37 Å.

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

    Science.gov (United States)

    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°.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Dan [Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Yamagata, Wataru; Kamei, Kaeko [Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Nozaki, Tomoyoshi [Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Harada, Shigeharu, E-mail: harada@kit.ac.jp [Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez, Héctor; Rivas, Blanca de las; Muñoz, Rosario [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Mancheño, José M., E-mail: xjosemi@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain)

    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.

  13. Cloning, crystallization and preliminary X-ray study of XC1258, a CN-hydrolase superfamily protein from Xanthomonas campestris

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Ying-Der; Chin, Ko-Hsin [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China); Shr, Hui-Lin [Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei,Taiwan (China); Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei,Taiwan (China); Gao, Fei Philip [National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310 (United States); Lyu, Ping-Chiang [Department of Life Science, National Tsing Hua University, Hsin-Chu,Taiwan (China); Wang, Andrew H.-J. [Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei,Taiwan (China); Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei,Taiwan (China); Chou, Shan-Ho, E-mail: shchou@nchu.edu.tw [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China)

    2006-10-01

    A CN-hydrolase superfamily protein from the plant pathogen X. campestris has been overexpressed in E. coli, purified and crystallized. CN-hydrolase superfamily proteins are involved in a wide variety of non-peptide carbon–nitrogen hydrolysis reactions, producing some important natural products such as auxin, biotin, precursors of antibiotics etc. These reactions all involve attack on a cyano or carbonyl carbon by a conserved novel catalytic triad Glu-Lys-Cys through a thiol acylenzyme intermediate. However, classification into the CN-hydrolase superfamily based on sequence similarity alone is not straightforward and further structural data are necessary to improve this categorization. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC1258, a CN-hydrolase superfamily protein from the plant pathogen Xanthomonas campestris (Xcc), are reported. The SeMet-substituted XC1258 crystals diffracted to a resolution of 1.73 Å. They are orthorhombic and belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 143.8, b = 154.63, c = 51.3 Å, respectively.

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

    Institute of Scientific and Technical Information of China (English)

    Yanfeng Zhang; Xiaoli Gao; Yi Zheng; R. Michae; Garavito

    2011-01-01

    Succinic semialdehyde reductase (SSAR) is an important enzyme involved in γ-aminobutyrate (GABA) metabolism.By converting succinic semialdehyde (SSA) to γ-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 coil 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+,and the resulting crystals diffracted to 1.89 (A) (GsSSAR) and 2.25 (A)(GmSSAR) resolution.The GsSSAR and GmSSAR crystals belong to the space groups P21221 (a =99.61 (A),b =147.49 (A),c =182.47 A) and P1 (a =75.97 (A) b =79.14 (A) c =95.47 (A),α =82.15°,β =88.80°,γ=87.66°),respectively.Preliminary crystallographic data analysis suggests the presence of eight protein monomers in the asymmetric units for both GsSSAR and GmSSAR.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

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

    Science.gov (United States)

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

    2015-12-01

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

  17. Cloning, expression and purification of orthologous membrane proteins: a general protocol for preparation of the histidine sensor kinase ETR1 from different species.

    Science.gov (United States)

    Classen, Elisa; Groth, Georg

    2012-03-01

    Orthologous proteins do not necessarily share the same function in all species and those sharing the same function might employ a modified catalytic mechanism. Thus, comparative analysis of homologous or orthologous proteins from different organisms can provide detailed information on the function and the mechanism of an entire protein family. The sensor kinase ETR1 from Arabidopsis thaliana has been well characterized by genetic, physiological and biochemical studies. However, as further model plants are coming into focus for plant hormone research, a general protocol for isolation and purification of orthologous ETR1 proteins seems instrumental for a detailed molecular analysis of this protein family. In this study, we describe the native purification of recombinant ETR1 from Arabidopsis thaliana by mild solubilization with the zwitter-ionic detergent Fos-Choline-14 and single-step purification by immobilized metal ion affinity chromatography. The same protocol was successfully applied for the purification of the orthologous proteins from the moss Physcomitrella patens subsp. patens and the tomato Lycopersicon esculentum. The successful transfer of the purification protocol to proteins of the same family which share sequence identity of 63-80% only suggests that this protocol presents a general purification strategy which is likely to apply also to the purification of other members of the sensor histidine kinase family.

  18. Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima

    Science.gov (United States)

    Ethayathulla, Abdul S.; Bessho, Yoshitaka; Shinkai, Akeo; Padmanabhan, Balasundaram; Singh, Tej P.; Kaur, Punit; Yokoyama, Shigeyuki

    2008-01-01

    Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6422, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å3 Da−1, which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan. PMID:18540059

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Material purification, crystal growth, and spectroscopy of Tm-doped KPb2Cl5 and KPb2Br5 for 2 μm photonic applications

    Science.gov (United States)

    Brown, E.; Kumi-Barimah, E.; Hömmerich, U.; Bluiett, A. G.; Trivedi, S. B.

    2014-05-01

    Results of the material purification, crystal growth, and optical characterization of Tm-doped KPb2Cl5 (KPC) and KPb2Br5 (KPB) for 2 μm photonic applications are presented. Undoped KPC and KPB were synthesized from ultra dry starting materials and purified through multi-pass translation in a zone-refinement system. 1 wt% of ultra-dry Tm-halides (TmCl3 or TmBr3) were added to the purified host materials and molten under bubbling of HCl or HBr gas, respectively. Tm: KPC and Tm: KPB crystals were subsequently grown using the Bridgman technique. The good optical quality of the resulting crystals was indicated by infrared transmissions as high as 70-75%. Under optical pumping, both crystals exhibited broad infrared emission bands centered at 1.82 μm with nearly exponential decay times ranging from 2-3 ms. Judd-Ofelt intensity parameters were calculated to determine the radiative lifetimes of the 3F4→3H6 Tm3+ transition. The optical absorption and emission cross-sections of Tm: KPC and Tm: KPB were determined using the McCumber theory. The investigated crystals were also evaluated as potential solid-state materials for optical cooling applications.

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

    Science.gov (United States)

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

    2014-02-01

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

  2. Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.

    Science.gov (United States)

    Hanania, Elie G; Fieck, Annabeth; Stevens, Janine; Bodzin, Leon J; Palsson, Bernhard Ø; Koller, Manfred R

    2005-09-30

    Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.

  3. Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants

    Directory of Open Access Journals (Sweden)

    Vassiliki I. Peppa

    2015-06-01

    Full Text Available SRL is a cell wall associated developmental-stage specific lectin secreted by Sclerotium rolfsii, a soil-born pathogenic fungus. SRL displays specificity for TF antigen (Galβ1→3GalNAc-α-Ser//Thr expressed in all cancer types and has tumour suppressing effects in vivo. Considering the immense potential of SRL in cancer research, we have generated two variant gene constructs of SRL and expressed in E. coli to refine the sugar specificity and solubility by altering the surface charge. SSR1 and SSR2 are two different recombinant variants of SRL, both of which recognize TF antigen but only SSR1 binds to Tn antigen (GalNAcα-Ser/Thr. The glycan array analysis of the variants demonstrated that SSR1 recognizes TF antigen and their derivative with high affinity similar to SRL but showed highest affinity towards the sialylated Tn antigen, unlike SRL. The carbohydrate binding property of SSR2 remains unaltered compared to SRL. The crystal structures of the two variants were determined in free form and in complex with N-acetylglucosamine at 1.7 Å and 1.6 Å resolution, respectively. Structural analysis highlighted the structural basis of the fine carbohydrate specificity of the two SRL variants and results are in agreement with glycan array analysis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Neil G., E-mail: neison@chem.gla.ac.uk; Riboldi-Tunnicliffe, Alan [Department of Chemistry and WestCHEM, Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom); Mitchell, Timothy J. [Division of Infection and Immunity (IBLS), Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom); Isaacs, Neil W. [Department of Chemistry and WestCHEM, Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

    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.

  5. Purification of Phosphoric Acid Hemihydrate Crystal by Dry-sweating%干法发汗提纯半水磷酸晶体的实验研究

    Institute of Scientific and Technical Information of China (English)

    王保明; 李军; 贾旭宏; 齐亚兵; 罗建洪

    2013-01-01

    采用干法发汗提纯半水磷酸晶体制备高纯磷酸.考察了发汗过程中发汗温度、冷却速率、酸浓度、发汗时间等参数对Mg和Sb脱除及净化效率的影响.结果表明:发汗温度越高,杂质脱除越快,净化效率越低,而且所得产品杂质含量也越少;结晶过程中的冷却速率越大,所得晶体中杂质含量也越多,杂质在发汗过程中脱除也越快,净化效率也越低,但是最终产品杂质含量越高;半水磷酸晶体中初始杂质含量越少,发汗所得产品纯度也越高,因此干法发汗可以用来精制磷酸.%A method of dry-sweating was applied to-purify phosphoric acid hemihydrate crystals to obtain high purity phosphoric acid. The effects of operating parameters, such as sweating temperature, cooling rate, phosphoric acid concentration and sweating time on the removal and purification efficiency of Mg and Sb were investigated. The experimental results showed that the content and purification efficiency of impurities decrease with the increase of sweating temperature, the content of impurities in the product obtained at higher sweating temperature are lower. The content of impurities in the crystals formed at higher cooling rate during crystallization are more, and the impurities can be removed easily during dry-sweating, moreover the purification efficiency of impurities are lower, however the content of impurities in the final product are more. The less the initial content of impurities in the crystals is, the higher the purity of product is. Therefore, dry-sweating can be used to fine phosphoric acid.

  6. Produção, purificação, clonagem e aplicação de enzimas líticas Production, purification, cloning and application of lytic enzymes

    Directory of Open Access Journals (Sweden)

    Luciana Francisco Fleuri

    2005-10-01

    Full Text Available Lytic enzymes such as beta-1,3 glucanases, proteases and chitinases are able to hydrolyse, respectively, beta-1,3 glucans, mannoproteins and chitin, as well as the cell walls of many yeast species. Lytic enzymes are useful in a great variety of applications including the preparation of protoplasts; the extraction of proteins, enzymes, pigments and functional carbohydrates; pre-treatment for the mechanical rupture of cells; degradation of residual yeast cell mass for the preparation of animal feed; analysis of the yeast cell wall structure and composition; study of the yeast cell wall synthesis and the control of pathogenic fungi. This review presents the most important aspects with respect to lytic enzymes, especially their production, purification, cloning and application.

  7. Separation and purification of dodecanedioic acid from its homologous compounds by falling film crystallization%应用降膜结晶法将十二烷二酸从同系物中分离及精制

    Institute of Scientific and Technical Information of China (English)

    李裕; 刘有智; 齐雪琴

    2004-01-01

    Separation and purification ofdodecanedioic acid(DDDA)from its homologous compounds were studied experimentally by falling film crystallization(FFC).The influences of various operation parameters,including crystallizing time,flow rate of melt and temperature of glycerine bath,on purity of DDDA and crystallizing rate were investigated.Over 99%(by mole)DDDA was obtained for a feed composition of 96%(by mole).The main factors affecting the separation efficiency are flow rate of melt and temperature of glycerine bath.The crystallizing layer of DDDA was further purified by sweating and blasting.A set of optimized operation data are provided for better understanding the mechanism of heat and mass transfer in FFC,and for further industrial application of DDDA purification process.

  8. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Gohain, Neelakshi [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany); Thomashow, Linda S. [Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430 (United States); USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, Pullman, Washington 99164-6430 (United States); Mavrodi, Dmitri V. [Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430 (United States); Blankenfeldt, Wulf, E-mail: wulf.blankenfeldt@mpi-dortmund.mpg.de [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rong, Hui; Li, Yan; Lou, Xiao-hua; Zhang, Xio; Gao, Yong-xiang; Teng, Mai-kun, E-mail: mkteng@ustc.edu.cn; Niu, Li-wen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Crystallization using reverse micelles and water-in-oil microemulsion systems: the highly selective tool for the purification of organic compounds from complex mixtures.

    Science.gov (United States)

    Kljajic, Alen; Bester-Rogac, Marija; Klobcar, Andrej; Zupet, Rok; Pejovnik, Stane

    2013-02-01

    The active pharmaceutical ingredient orlistat is usually manufactured using a semi-synthetic procedure, producing crude product and complex mixtures of highly related impurities with minimal side-chain structure variability. It is therefore crucial for the overall success of industrial/pharmaceutical application to develop an effective purification process. In this communication, we present the newly developed water-in-oil reversed micelles and microemulsion system-based crystallization process. Physiochemical properties of the presented crystallization media were varied through surfactants and water composition, and the impact on efficiency was measured through final variation of these two parameters. Using precisely defined properties of the dispersed water phase in crystallization media, a highly efficient separation process in terms of selectivity and yield was developed. Small-angle X-ray scattering, high-performance liquid chromatography, mass spectrometry, and scanning electron microscopy were used to monitor and analyze the separation processes and orlistat products obtained. Typical process characteristics, especially selectivity and yield in regard to reference examples, were compared and discussed. Copyright © 2012 Wiley Periodicals, Inc.

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

    Energy Technology Data Exchange (ETDEWEB)

    Huché, Frédéric, E-mail: huche@pasteur.fr [Fachbereich Biologie, Universität Konstanz, 78457 Konstanz (Germany); Unité des Membranes Bactériennes, CNRS URA 2172, Département de Microbiologie Fondamentale et Médicale, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris CEDEX 15 (France); Delepelaire, Philippe; Wandersman, Cécile [Unité des Membranes Bactériennes, CNRS URA 2172, Département de Microbiologie Fondamentale et Médicale, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris CEDEX 15 (France); Welte, Wolfram [Fachbereich Biologie, Universität Konstanz, 78457 Konstanz (Germany)

    2006-01-01

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

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

    Science.gov (United States)

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

    2009-03-01

    Transforming growth factor beta-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.

  14. Purification, crystallization and preliminary X-ray analysis of the membrane-bound [NiFe] hydrogenase from Allochromatium vinosum

    Energy Technology Data Exchange (ETDEWEB)

    Kellers, Petra; Ogata, Hideaki; Lubitz, Wolfgang, E-mail: lubitz@mpi-muelheim.mpg.de [Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, D-45470 Mülheim an der Ruhr (Germany)

    2008-08-01

    This article describes the first successful crystallization of a membrane-bound [NiFe] hydrogenase isolated from a photosynthetic organism (A. vinosum). The crystals obtained produced diffraction patterns up to 2.5 Å resolution. The membrane-bound [NiFe] hydrogenase is a unique metalloprotein that is able to catalyze the reversible oxidation of hydrogen to protons and electrons during a complex reaction cycle. The [NiFe] hydrogenase was isolated from the photosynthetic purple sulfur bacterium Allochromatium vinosum and its crystallization and preliminary X-ray analysis are reported. It was crystallized by the hanging-drop vapour-diffusion method using sodium citrate and imidazole as crystallization agents. The crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 205.00, b = 217.42, c = 120.44 Å. X-ray diffraction data have been collected to 2.5 Å resolution.

  15. Purification, crystallization and preliminary X-ray diffraction analysis of an oomycete-derived Nep1-­like protein

    Science.gov (United States)

    Luberacki, Borries; Weyand, Michael; Seitz, Ulrich; Koch, Wolfgang; Oecking, Claudia; Ottmann, Christian

    2008-01-01

    The elicitor protein Nep1-like protein from the plant pathogen Pythium aphanidermatum was purified and crystallized using the hanging-drop vapour-diffusion method. A native data set was collected to 1.35 Å resolution at 100 K using synchrotron radiation. Since selenomethionine-labelled protein did not crystallize under the original conditions, a second crystal form was identified that yielded crystals that diffracted to 2.1 Å resolution. A multiple-wavelength anomalous dispersion (MAD) experiment was performed at 100 K and all four selenium sites were identified, which allowed solution of the structure. PMID:19052381

  16. Purification, crystallization and preliminary X-ray diffraction analysis of an oomycete-derived Nep1-like protein.

    Science.gov (United States)

    Luberacki, Borries; Weyand, Michael; Seitz, Ulrich; Koch, Wolfgang; Oecking, Claudia; Ottmann, Christian

    2008-12-01

    The elicitor protein Nep1-like protein from the plant pathogen Pythium aphanidermatum was purified and crystallized using the hanging-drop vapour-diffusion method. A native data set was collected to 1.35 A resolution at 100 K using synchrotron radiation. Since selenomethionine-labelled protein did not crystallize under the original conditions, a second crystal form was identified that yielded crystals that diffracted to 2.1 A resolution. A multiple-wavelength anomalous dispersion (MAD) experiment was performed at 100 K and all four selenium sites were identified, which allowed solution of the structure.

  17. Cloning, expression, crystallization and preliminary X-ray analysis of the XMT and DXMT N-methyltransferases from Coffea canephora (robusta)

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, Andrew A., E-mail: andrewmc@embl.fr [European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble (France); Biget, Laurent [Nestlé Research and Development, 101 Avenue Gustave Eiffel, Notre-Dame D’Oe, 37097 Tours (France); Lin, Chenwei [Department of Plant Breeding and Genetics, Department of Plant Biology, Cornell University, Ithaca, NY 14853 (United States); Petiard, Vincent [Nestlé Research and Development, 101 Avenue Gustave Eiffel, Notre-Dame D’Oe, 37097 Tours (France); Tanksley, Steve D. [Department of Plant Breeding and Genetics, Department of Plant Biology, Cornell University, Ithaca, NY 14853 (United States); McCarthy, James G. [Nestlé Research and Development, 101 Avenue Gustave Eiffel, Notre-Dame D’Oe, 37097 Tours (France); European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble (France)

    2007-04-01

    The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). Caffeine is a secondary metabolite produced by a variety of plants including Coffea canephora (robusta) and there is growing evidence that caffeine is part of a chemical defence strategy protecting young leaves and seeds from potential predators. The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). The crystals are orthorhombic, with space group P2{sub 1}2{sub 1}2{sub 1} for XMT and C222{sub 1} for DXMT. X-ray diffraction to 2.8 Å for XMT and to 2.5 Å for DXMT have been collected on beamline ID23-1 at the ESRF.

  18. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yang-De, E-mail: zhangyd1960@yahoo.com.cn; Li, Hao [National Hepatobiliary and Enteric Surgery Research Center of The Ministry of Health, Xiangya Hospital, Central South University, Hunan Province (China); Liu, Hui; Pan, Yi-Feng [Biochemistry Laboratory, Institution of Biomedical Engineering, Central South University, Hunan Province (China); National Hepatobiliary and Enteric Surgery Research Center of The Ministry of Health, Xiangya Hospital, Central South University, Hunan Province (China)

    2007-02-01

    Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2{sub 1}2{sub 1}2{sub 1} and tetragonal P4{sub 1}2{sub 1}2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*{sub 65–224} structure was determined by molecular replacement.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki, E-mail: kunisima@spring8.or.jp

    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.

  20. Purification, crystallization and preliminary X-ray diffraction of the G3BP1 NTF2-like domain

    DEFF Research Database (Denmark)

    Vognsen, Tina; Möller, Ingvar Rúnar; Kristensen, Ole

    2011-01-01

    The nuclear transport factor 2-like (NTF2-like) domain of human G3BP1 was subcloned, overexpressed in Escherichia coli and purified. Crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected to 3.6 Å resolution using synchrotron radiation. The crystals...

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

    Energy Technology Data Exchange (ETDEWEB)

    Ohtsuki, Takayuki [Department of Bimolecular Science, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510 (Japan); Ohshima, Shigeru [Department of Environmental Science, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510 (Japan); Uchida, Akira, E-mail: auchida@biomol.sci.toho-u.ac.jp [Department of Bimolecular Science, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510 (Japan)

    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.

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

    Science.gov (United States)

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

    2014-02-10

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

  3. Gene Cloning, Expression, Purification and Antigenicity Identification of Tamm-Horsfall Protein Domain%Tamm-Horsfall蛋白片段的基因克隆、表达、纯化及抗原性鉴定

    Institute of Scientific and Technical Information of China (English)

    朱美财; 马红雨; 王红; 陈涛

    2012-01-01

    目的 克隆并表达尿调节素(Tamm-Horsfall,THP)蛋白片段的基因,检测其抗原性.方法 利用RT-PCR技术扩增THP蛋白片段基因,测序后将目的基因克隆入原核表达质粒pET28a载体,转化至大场杆菌BL21(DE3),经IPTG诱导表达,获得重组THP蛋白片段.经NI亲和层析柱纯化,用western-blot检测重组蛋白与THP单克隆抗体结合活性.结果 克隆THP蛋白片段基因,其开放阅读框为195 bp编码65个氨基酸.表达的重组THP蛋白片段以包涵体形式存在,菌体蛋白纯化后,经western-blot检测具有较好的抗原性.结论 成功地克隆和表达了THP片段的基因,为抗THP单克隆抗体制备和THP检测试剂盒的研发奠定了基础.%Objective To investigate the cloning and expression of a part of gene encoding Tamm- Horsfall protein (THP) domain and exam its antigenicity. Methods A part of gene encoding Tamm-Horsfall protein domain was amplified by reverse-translation polymerase chain reaction (RT-PCR). After sequencing, the intent gene was cloned into expression plasmid vector pET28a and then transformed into Ecoli. BL21 (DE3). The THP domain was expressed by inducing with IPTG, purified by Ni-affinity chromatography. The combination activity of re-combinant protein with anti-THP monoclonal antibody was detected by western-blot. Results A 195 bp fragment from THP gene encoding 65 amino acids was cloned. The THP domains were expressed as inclusion bodies. After purification, it could be recognized by anti-THP monoclonal antibody by western-blot. Conclusion A part of gene encoding THP domain was successfully cloned and expressed. It could be used to develop anti-THP monoclonal antibody and THP test kit.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, Sarvind Mani; Ramachandran, Ravishankar, E-mail: r-ravishankar@cdri.res.in [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226001 (India)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bagautdinov, Bagautdin [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Sugahara, Mitsuaki [Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Kunishima, Naoki, E-mail: kunisima@spring8.or.jp [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

    2007-01-01

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

  6. Expression, Purification, Crystallization and Molecular Replacement Studies of TorI, an Inhibition Protein of Tor System

    Institute of Scientific and Technical Information of China (English)

    HUANG Wei; YUAN Cai; Ansaldi Mireille; Morelli Xavier; Edward J. Meehan; CHEN Li-Qing; HUANG Ming-Dong

    2007-01-01

    TorI, a Tor system inhibitor acting through protein-protein interaction with the TorR response regulator, is an excisionase that interacts with the integrase and DNA during prophage excision. It has been crystallized by the vapor-diffusion method using polyethylene glycol 3350 as a precipitant at pH 8.5. The X-ray diffraction data sets from the TorI crystal was collected at a resolution of 2.1 (A), using a synchrotron source. The crystal belongs to primitive monoclinic lattice with cell parameters of 46.210(A) × 53.992(A) × 73.561(A)

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

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi-Hung [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Peng, Wen-Yan [Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Huang, Yen-Chieh [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Guan, Hong-Hsiang; Hsieh, Ying-Cheng [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Liu, Ming-Yih [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Chang, Tschining [Department of Hospitality Management, Nan Jeon Institute of Technology, Yen-Shui, Tainan 73746,Taiwan (China); Chen, Chun-Jung, E-mail: cjchen@nsrrc.org.tw [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Department of Physics, National Tsing-Hua University, Hsinchu 30013,Taiwan (China)

    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%.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chung-Der; Huang, Tien-Feng [Department of Physics, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Lin, Chih-Hao [Institute of Biological Chemistry, National Taiwan University, Taipei 110,Taiwan (China); Guan, Hong-Hsiang; Hsieh, Yin-Cheng [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Lin, Yi-Hung; Huang, Yen-Chieh; Liu, Ming-Yih [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Chang, Wen-Chang, E-mail: wchang@ntu.edu.tw [Institute of Biological Chemistry, National Taiwan University, Taipei 110,Taiwan (China); Chen, Chun-Jung, E-mail: wchang@ntu.edu.tw [Department of Physics, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China)

    2007-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Shlegel V.N.

    2014-01-01

    Full Text Available 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.

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

    CERN Document Server

    Shlegel, V N; 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

    2013-01-01

    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.

  11. Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum

    Energy Technology Data Exchange (ETDEWEB)

    Byrnes, Laura J. [Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025 (United States); Badarau, Adriana; Vakulenko, Sergei B. [Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 (United States); Smith, Clyde A., E-mail: csmith@slac.stanford.edu [Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025 (United States)

    2008-02-01

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

  12. Production, Purification, and Gene Cloning of a β-Fructofuranosidase with a High Inulin-hydrolyzing Activity Produced by a Novel Yeast Aureobasidium sp. P6 Isolated from a Mangrove Ecosystem.

    Science.gov (United States)

    Jiang, Hong; Ma, Yan; Chi, Zhe; Liu, Guang-Lei; Chi, Zhen-Ming

    2016-08-01

    After screening of over 300 yeast strains isolated from the mangrove ecosystems, it was found that Aureobasidium sp. P6 strain had the highest inulin-hydrolyzing activity. Under the optimal conditions, this yeast strain produced an inulin-hydrolyzing activity of 30.98 ± 0.8 U/ml after 108 h of a 10-l fermentation. After the purification, a molecular weight of the enzyme which had the inulin-hydrolyzing activity was estimated to be 47.6 kDa, and the purified enzyme could actively hydrolyze both sucrose and inulin and exhibit a transfructosylating activity at 30.0 % sucrose, converting sucrose into fructooligosaccharides (FOS), indicating that the purified enzyme was a β-D-fructofuranosidase. After the full length of a β-D-fructofuranosidase gene (accession number KU308553) was cloned from Aureobasidium sp. P6 strain, a protein deduced from the cloned gene contained the conserved sequences MNDPNGL, RDP, ECP, FS, and Q of a glycosidehydrolase GH32 family, respectively, but did not contain a conserved sequence SVEVF, and the amino acid sequence of the protein from Aureobasidium sp. P6 strain had a high similarity to that of the β-fructofuranosidase from any other fungal strains. After deletion of the β-D-fructofuranosidase gene, the disruptant still had low inulin hydrolyzing and invertase activities and a trace amount of the transfructosylating activity, indicating that the gene encoding an inulinase may exist in the Aureobasidium sp. P6 strain.

  13. Overexpression, purification and crystallization of lysine ∊-aminotransferase (Rv3290c) from Mycobacterium tuberculosis H37Rv

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, Sarvind Mani; Ramachandran, Ravishankar, E-mail: ravi-anitha@yahoo.com [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226001 (India)

    2006-06-01

    Lysine ∊-aminotransferase from M. tuberculosis has been crystallized. Preliminary crystallographic analysis shows that there is one monomer in the asymmetric unit of the crystal. Lysine ∊-aminotransferase (LAT) is a protein involved in lysine catabolism; it belongs to the aminotransferase family of enzymes, which use pyridoxal 5′-phosphate (PLP) as a cofactor. LAT probably plays a significant role during the persistent/latent phase of Mycobacterium tuberculosis, as observed by its up-regulation by ∼40-fold during this stage. Crystals of recombinant LAT have been grown in 0.1 M trisodium citrate dihydrate solution containing 0.2 M ammonium acetate and 25% PEG 4000 in the pH range 5.4–6.0. Diffraction data extending to 1.98 Å were collected at room temperature from a single crystal. Crystals are trigonal in shape and belong to space group P3{sub 1}21, with unit-cell parameters a = 103.26, b = 103.26, c = 98.22 Å. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V{sub M}) of 3.1 Å{sup 3} Da{sup −1}.

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

    Energy Technology Data Exchange (ETDEWEB)

    Byrnes, L.J.; /SLAC, SSRL; Badarau, A.; Vakulenko, S.B.; /Notre Dame U.; 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.

  15. Why Clone?

    Science.gov (United States)

    ... How might cloning be used in medicine? Cloning animal models of disease Much of what researchers learn ... issue of the genetic reshuffling that happensduring sexual reproduction and simply clone our drug-producing cow. Cloning ...

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

    Science.gov (United States)

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Gonçalves, A. M. D.; Rêgo, A. T. [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal); Thomaz, M. [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal); Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2748-901 Oeiras (Portugal); Enguita, F. J., E-mail: fenguita@fm.ul.pt [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal); Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa (Portugal); Carrondo, M. A. [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi-Hung [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Li, Hsin-Tai [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Huang, Yen-Chieh [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Hsieh, Ying-Cheng; Guan, Hong-Hsiang [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Liu, Ming-Yih [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Chang, Tschining; Wang, Andrew H.-J., E-mail: ahjwang@gate.sinica.edu.tw [Institute of Biological Chemistry, Academia Sinica, Taipei 11529,Taiwan (China); Chen, Chun-Jung, E-mail: ahjwang@gate.sinica.edu.tw [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Department of Physics, National Tsing-Hua University, Hsinchu 30013,Taiwan (China)

    2006-06-01

    Rice Bowman–Birk inhibitor was expressed and crystallized. Bowman–Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 Å, the unit cell belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 74.37, b = 96.69, c = 100.36 Å. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, SeungBum [Department of Biological and Molecular Engineering, College of Engineering, Ajou University, Suwon (Korea, Republic of); Joo, Sangbum [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon (Korea, Republic of); Yoon, Hyun C.; Ryu, Yeonwoo [Department of Biological and Molecular Engineering, College of Engineering, Ajou University, Suwon (Korea, Republic of); Kim, Kyeong Kyu, E-mail: kkim@med.skku.ac.kr [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon (Korea, Republic of); Kim, T. Doohun, E-mail: kkim@med.skku.ac.kr [Department of Biological and Molecular Engineering, College of Engineering, Ajou University, Suwon (Korea, Republic of)

    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°.

  1. Expression, purification, crystallization and X-ray diffraction studies of the molecular chaperone prefoldin from Homo sapiens.

    Science.gov (United States)

    Aikawa, Yoshiki; Kida, Hiroshi; Nishitani, Yuichi; Miki, Kunio

    2015-09-01

    Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7 Å resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 123.2, b = 152.4, c = 105.9 Å.

  2. Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor.

    Science.gov (United States)

    Sanchez, J F; Hoh, F; Strub, M P; Strub, J M; Van Dorsselaer, A; Lehrer, R; Ganz, T; Chavanieu, A; Calas, B; Dumas, C; Aumelas, A

    2001-11-01

    Numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence which belongs to the cathelicidin family of proteins. The three-dimensional structure of this cathelicidin motif, which contains two disulfide bonds, has not yet been reported. The cathelicidin motif (ProS) of the protegrin-3 precursor was overexpressed in Escherichia coli as a His-tagged protein. The His(6) tag was removed by thrombin cleavage. ProS was purified to homogeneity and single crystals were obtained by the hanging-drop vapour-diffusion method at pH 3-4. Preliminary X-ray diffraction analysis indicated that these crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 51.42, c = 134.25 A. These crystals diffracted beyond 2.75 A (1.9 A at ESRF) and contain one molecule per asymmetric unit.

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

    Science.gov (United States)

    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.

  4. Purification, crystallization and preliminary X-ray crystallographic analysis of human ppGpp hydrolase, Mesh1

    Directory of Open Access Journals (Sweden)

    Dawei Sun

    2010-09-01

    Full Text Available Bacterial SpoT is a Mn2+-dependent pyrophosphohydrolase to hydrolyze guanosine 3’-diphosphate-5’-diphosphate to guanosine diphosphate and pyrophosphate. In this study, SpoT ortholog from Homo sapiens (hMesh1, was over-expressed in Escherichia coli, purified and crystallized using hanging-drop vapour-diffusion method with polyethylene glycol and sodium citrate. The native crystal of hMesh1 was diffracted to 2.1 Å using a synchrotron-radiation source and belonged to the monoclinic space group P21 with cell dimensions of a = 53.27 Å, b = 62.61 Å, c = 52.45 Å and β= 94.96˚. The crystal contains two molecules in the asymmetric unit, with a solvent content of 44% and a Matthews coefficient VM value of 2.18 Å3/Da.

  5. Cloning,expression and purification of the ligand-binding region of human IL-6R in E.coli and its preliminary functional identification

    Institute of Scientific and Technical Information of China (English)

    段聚宝; 王嘉玺; 韩家淮; 彭善云; 邹民吉; 苗继红; 赵春文; 马贤凯

    1995-01-01

    The ligand-binding region of human IL-6R is taken as the target gene fragment to be clonedand expressed.With pET-3b as expressing vector,two recombinants pET-6R(B)and pET-6R(B)4 have beenconstructed encoding the ligand-binding region(28 kD)of hIL-6R and its dimmer(53 kD),respectively.Afterinduction with IPTG,they produced two proteins rIL6R-28 of 28 kD and rIL6R-53 of 53 kD amounting to 50%and 30% of total bacteria proteins,respectively.The expressed products were mainly recovered as inclusionbodies.After purification and renaturation,both of them were capable of augmenting the growth-stimulatingeffect of IL-6 on 7TD1 cells,an IL-6 dependent cell line.The result of ELISA also revealed that bothrIL6R-28 and rIL6R-53 had the obvious ligand-binding activity.

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

    Science.gov (United States)

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

    2004-04-01

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

  7. Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli.

    Science.gov (United States)

    Ramesh, M V; Podkovyrov, S M; Lowe, S E; Zeikus, J G

    1994-01-01

    The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.

  8. Amplification, cloning and expression of Brucella melitensis bp26 gene (OMP28 isolated from Markazi province (Iran and purification of Bp26 Protein

    Directory of Open Access Journals (Sweden)

    Hosseini, S.D.

    2013-12-01

    Full Text Available Brucellosis is a debilitative disease that imposes costs on both economy and society. It is shown that although the vaccine can prevent abortion, it does not provide complete protection against infection. In Iran, Brucella melitensis is a common causative agent for brucellosis and BP26 protein of this bacterium having a good antigenesity and an important vaccine candidate. In this study B. melitensis bp26 gene was cloned first in to PTZ57R/T vector and accessed on the PET28a vector and sequenced. Recombinant vector transformed and expressed in to E. coli BL21 (DE3 and then recombinant protein was purified with Ni-NTA column of chromatography against His tag. Obtained rOmp28 could be used as a research experimental tool to find its potential as a detection kit and vaccine candidate.

  9. Purification, characterization and partial cDNA cloning of high-temperature stress-induced protein from French bean (Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Nagesh Babu, R.

    2013-02-01

    Full Text Available In order to identify the components of high temperature response in French bean, three heat shock proteins induced under high temperature were purified to homogeneity by Carboxy methyl cellulose and sephadex G-100 chromatography followed by preparative SDS-PAGE. Two of these, Hsp1 and Hsp3 were further characterized by immuno-detection with polyclonal antibodies. Hsp3 exhibited ATPase and chaperone activity with malate dehydrogenase and citrate synthase. Partial cDNA for Hsp3 synthesized using the primer derived from amino-terminal sequence was cloned and expressed in Escherichia coli. The recombinant protein possesses ATPase activity, and showed thermal protection at 50°C in Escherichia coli. The translated partial cDNA showed homology with stress induced proteins including ATPases from higher plants. These results supported the fact that French bean response to high temperature stress involves Hsps as one of the principal components.

  10. Cloning, expression, and purification of the His(6)-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Ahring, Birgitte Kiær

    2003-01-01

    The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E coli BL21(DE3)pLysS and E. coli Rosetta(DE3)p......LysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni2+-IDA-Sepharose, dialyzed...

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

    Science.gov (United States)

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

    2006-04-01

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

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Coelho, Catarina; González, Pablo J.; Trincão, José; Carvalho, Ana L.; Najmudin, Shabir [REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Hettman, Thomas; Dieckman, Stephan [Institute for Molecular Biotechnology, Beutenbergstrasse 11, 07745 Jena (Germany); Moura, José J. G.; Moura, Isabel; Romão, Maria J., E-mail: mromao@dq.fct.unl.pt [REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal)

    2007-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Garnett, James A.; Diallo, Mamou; Matthews, Steve J., E-mail: s.j.matthews@imperial.ac.uk [Imperial College London, South Kensington, London SW7 2AZ (United Kingdom)

    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

  15. cDNA Cloning, Overexpression, Purification and Pharmacologic Evaluation for Anticancer Activity of Ribosomal Protein L23A Gene (RPL23A from the Giant Panda

    Directory of Open Access Journals (Sweden)

    Si-Nan Zhang

    2012-02-01

    Full Text Available RPL23A gene encodes a ribosomal protein that is a component of the 60S subunit. The protein belongs to the L23P family of ribosomal proteins, which is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of ribosomal protein L23A (RPL23A gene of the Giant Panda (Ailuropoda melanoleuca. The cDNA of RPL23A was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL23A cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. Recombinant protein of RPL23A obtained from the experiment acted on Hep-2 cells and human HepG-2 cells, then the growth inhibitory effect of these cells was observed by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide assay. The result indicated that the length of the fragment cloned is 506 bp, and it contains an open-reading frame (ORF of 471 bp encoding 156 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL23A protein is 17.719 kDa with a theoretical pI 11.16. The molecular weight of the recombinant protein RPL23A is 21.265 kDa with a theoretical pI 10.57. The RPL23A gene can be really expressed in E. coli and the RPL23A protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 22 KDa polypeptide. The data showed that the recombinant protein RPL23A had a time- and dose-dependency on the cell growth inhibition rate. The data also indicated that the effect at low concentrations was better than at high concentrations on Hep-2 cells, and that the concentration of 0.185 μg/mL had the best rate of growth inhibition of 36.31%. All results of the experiment revealed that the recombinant protein RPL23A exhibited anti-cancer function on the Hep-2 cells. The study provides a scientific basis and aids

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

    Science.gov (United States)

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

    2009-07-01

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

  17. Purification, crystallization and preliminary crystallographic analysis of the SpaA backbone-pilin subunit from probiotic Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Singh, Deepak; von Ossowski, Ingemar; Palva, Airi; Krishnan, Vengadesan

    2013-10-01

    Lactobacillus rhamnosus GG, a widely used Gram-positive probiotic strain, is clinically well known for its perceived health-promoting effects. It has recently been shown to display proteinaceous pilus fibres (called SpaCBA) on its cell surface. Structurally, SpaCBA pili possess a characteristic three-pilin polymerized architecture, with repeating SpaA major pilins that form the backbone and two types of minor subunits (SpaB and SpaC). In this study, recombinant SpaA protein was purified, characterized and crystallized. The crystals diffracted to a resolution of 2.0 Å and belonged to space group C2, with unit-cell parameters a=227.9, b=63.2, c=104.3 Å, β=95.1°.

  18. 重组内肽酶AspN的原核表达纯化和活性鉴定%Cloning,Expression,Purification and Characterization of Soluble Recombinant Endoproteainase AspN

    Institute of Scientific and Technical Information of China (English)

    郑娟; 郭怀祖; 李晶; 段树燕; 赵自叶; 张大鹏; 郭尚敬; 王皓

    2015-01-01

    Endoproteinase AspN(flavastacin)is a zinc metalloendopeptidase which can selectively cleave peptide bonds N-terminal to aspartic acid residues, also it is one of most widely used proteolytic enzyme used for protein digestion prior to MS or HPLC analysis. The natural endoproteinase AspN was extract from the bacteria,Elizabethkingia meningoseptica. Some factors including low yields, difficulty in purification and high cost, greatly limit the application of the enzyme. Target gene was cloned into an pET32a expression vector and transformed into E.coli BL21(DE3). This is the first report for the soluble expression of AspN-Trx in prokaryotic expression system and a poly-histidine tag enabled purification by Ni affinity chromatography. The activity of the recombinant endopeptidase AspN was identified by HPLC, SDS-PAGE and the fluorogenic substrate Anthranilyl-Ala-Phe-Ala-Phe-Asp-Val-Phe(NO2)-Tyr-Asp. The results showed that the recombinant endopeptidase AspN was consistent with the standard enzyme and can be used widely.%蛋白内肽酶AspN是一种锌金属内切肽酶,能选择性切割天冬氨酸的N端肽键,广泛应用于蛋白的多肽制备及质量肽图谱鉴定。目前内肽酶AspN来源于细菌分泌,产量低,制备困难,成本高,极大地限制了该酶的应用。将脑膜脓毒性黄杆菌分泌的蛋白内肽酶AspN所对应的基因克隆入表达载体pET32a,导入E.coli BL21(DE3),首次运用原核表达系统进行可溶性融合表达,亲和层析对重组蛋白进行纯化。用HPLC、SDS-PAGE和荧光底物Anthranilyl-Ala-Phe-Ala-Phe-Asp-Val-Phe(NO2)-Tyr-Asp对重组酶进行酶活鉴定。结果表明重组的内肽酶AspN具有与标准品基本一致的酶切活性,能够较好地应用于生物和制药领域。

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

    Science.gov (United States)

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

    2015-06-01

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

  20. Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Wenqing; Zhao, Wei [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Wang, Xiaoping [National Conservation of Snake Island and Laotieshan Mountain, Dalian, Liaoning, 116041 (China); Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Dongwen; Sun, Jianping; Zhao, Wei; Zhang, Xiao; Shi, Yunyu; Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Dong, Yuhui; Liu, Peng [Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, 19B Yuquan Road, Beijing 100039 (China); Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    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.

  2. Isolation, purification, crystallization and preliminary crystallographic studies of sagitoxin, an oligomeric cardiotoxin from the venom of Naja naja saggitifera

    Science.gov (United States)

    Mir, Rafia; Sinha, Mau; Sharma, Sujata; Singh, Nagendra; Kaur, Punit; Srinivasan, A.; Singh, Tej P.

    2008-01-01

    Sagitoxin, a novel cardiotoxin from the venom of Naja naja saggitifera, has been successfully isolated, purified to homogeneity and crystallized. The toxin was purified using successive separation steps on a CM-Sephadex C-50 column and a reverse-phase column. The 6.75 kDa toxin was sequenced by the Edman method using a PPSQ-21 protein sequencer. It was crystallized using the hanging-drop vapour-diffusion method. The hexagonal-shaped crystals diffracted to 3.0 Å resolution and belonged to space group P64, with unit-cell parameters a = b = 111.1, c = 137.3 Å, γ = 120°. There are 36 molecules in the unit cell, which has an approximate solvent content of 80%. Structure determination revealed that the molecules of sagitoxin associate in a hexameric form and create a pore in the centre which has functional significance. PMID:18540072

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

    Science.gov (United States)

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

    2007-01-01

    The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe–4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 Å at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 Å, β = 100.7°, space group C2, and one heterodimer is present per asymmetric unit. PMID:17554176

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

    Energy Technology Data Exchange (ETDEWEB)

    Bahl, C.; MacEachran, D; O& apos; 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.

  5. Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Su In; Kim, Hee Sook [Kyungsung Univ., Busan (Korea, Republic of). Dept. of Food Science and Biotechnology; Choi, Sung Hee; Lee, Eun Yeol [Kyung Hee Univ., Gyeonggi-do (Korea, Republic of). Dept. of Chemical Engineering

    2012-09-15

    A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40 C. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M-G linkage than that in G-M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate. (orig.)

  6. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

    Science.gov (United States)

    Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

    2000-03-01

    Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

  7. Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities. Gene cloning, purification, and characterization of a novel pyridoxal enzyme.

    Science.gov (United States)

    Mihara, H; Kurihara, T; Yoshimura, T; Soda, K; Esaki, N

    1997-09-05

    Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine. Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.

  8. Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

    Science.gov (United States)

    Goncharov, I; Palfi, Z; Bindereif, A; Michaeli, S

    1999-04-30

    Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

  9. Purification, gene cloning, and biochemical characterization of a β-glucosidase capable of hydrolyzing sesaminol triglucoside from Paenibacillus sp. KB0549.

    Directory of Open Access Journals (Sweden)

    Arun Nair

    Full Text Available The triglucoside of sesaminol, i.e., 2,6-O-di(β-D-glucopyranosyl-β-D- glucopyranosylsesaminol (STG, occurs abundantly in sesame seeds and sesame oil cake and serves as an inexpensive source for the industrial production of sesaminol, an anti-oxidant that displays a number of bioactivities beneficial to human health. However, STG has been shown to be highly resistant to the action of β-glucosidases, in part due to its branched-chain glycon structure, and these circumstances hampered the efficient utilization of STG. We found that a strain (KB0549 of the genus Paenibacillus produced a novel enzyme capable of efficiently hydrolyzing STG. This enzyme, termed PSTG, was a tetrameric protein consisting of identical subunits with an approximate molecular mass of 80 kDa. The PSTG gene was cloned on the basis of the partial amino acid sequences of the purified enzyme. Sequence comparison showed that the enzyme belonged to the glycoside hydrolase family 3, with significant similarities to the Paenibacillus glucocerebrosidase (63% identity and to Bgl3B of Thermotoga neapolitana (37% identity. The recombinant enzyme (rPSTG was highly specific for β-glucosidic linkage, and k cat and k cat/K m values for the rPSTG-catalyzed hydrolysis of p-nitrophenyl-β-glucopyraniside at 37°C and pH 6.5 were 44 s(-1 and 426 s(-1 mM(-1, respectively. The specificity analyses also revealed that the enzyme acted more efficiently on sophorose than on cellobiose and gentiobiose. Thus, rPSTG is the first example of a β-glucosidase with higher reactivity for β-1,2-glucosidic linkage than for β-1,4- and β-1,6-glucosidic linkages, as far as could be ascertained. This unique specificity is, at least in part, responsible for the enzyme's ability to efficiently decompose STG.

  10. A cold-adapted carbohydrate esterase from the oil-degrading marine Bacterium Microbulbifer thermotolerans DAU221: gene cloning, purification, and characterization.

    Science.gov (United States)

    Lee, Yong-Suk; Heo, Jae Bok; Lee, Je-Hoon; Choi, Yong-Lark

    2014-07-01

    A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the α/β-class of protein consisted of a central six-stranded β-sheet flanked by eight α-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were 15°C and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50°C or 60°C for 6 h, and 89% of its enzyme activity when preincubated at 70°C for 1h . The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by Na(+) and Mg(2+) ions but was strongly inhibited by Cu(+) and Hg(2+) ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a Km of 0.278 mM and a kcat of 1.9 s(-1). Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

  11. Purification, gene cloning and characterization of an acidic β-1,4-glucanase from Phialophora sp. G5 with potential applications in the brewing and feed industries.

    Science.gov (United States)

    Zhao, Junqi; Shi, Pengjun; Yuan, Tiezheng; Huang, Huoqing; Li, Zhongyuan; Meng, Kun; Yang, Peilong; Yao, Bin

    2012-10-01

    An extracellular β-1,4-glucanase (CelG5, ∼55.0 kDa) was isolated from the culture filtrate of Phialophora sp. G5, and its encoding gene was cloned. The deduced amino acid sequence of CelG5 was at most 73.6% and 44.0%, respectively, identical with a hypothetical protein from Sordaria macrospora and an experimentally verified GH 7 endo-β-1,4-glucanase of Neurospora tetrasperma FGSC 2508. Native CelG5 had pH and temperature optima of pH 4.5-5.0 and 55-60°C. The enzyme showed some properties superior than most fungal β-1,4-glucanases, such as high activity over a wide pH range (exhibiting >50% of the maximum activity at pH 2.0-7.0), excellent stability in extreme acidic to alkaline conditions (pH 2.0-9.0), and strong resistance against pepsin and trypsin (retaining 89% and 94% activity, respectively). Recombinant CelG5 produced in Pichia pastoris had a molecular mass and a pH optimum similar to native CelG5, but with maximal activity at 65°C. Application tests showed that native CelG5 was stable under simulated gastric conditions (retaining >70% activity), and had capacity to decrease the viscosity of barley-bean feed (8.9% by 200 U CelG5) and mash (6.1% by 50 U CelG5) and increase the filtration rate of mash (18.4% by 50 U CelG5). These properties make CelG5 a good candidate for utilization in the animal feed and brewing industries.

  12. A new cold-adapted β-D-galactosidase from the Antarctic Arthrobacter sp. 32c – gene cloning, overexpression, purification and properties

    Directory of Open Access Journals (Sweden)

    Kur Józef

    2009-07-01

    Full Text Available Abstract Background The development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production. Results In this article, we present a new β-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this β-D-galactosidase has been isolated from the genomic DNA library of Antarctic bacterium Arthrobacter sp. 32c, sequenced, cloned, expressed in Escherichia coli and Pichia pastoris, purified and characterized. 27 mg of β-D-galactosidase was purified from 1 L of culture with the use of an intracellular E. coli expression system. The protein was also produced extracellularly by P. pastoris in high amounts giving approximately 137 mg and 97 mg of purified enzyme from 1 L of P. pastoris culture for the AOX1 and a constitutive system, respectively. The enzyme was purified to electrophoretic homogeneity by using either one step- or a fast two step- procedure including protein precipitation and affinity chromatography. The enzyme was found to be active as a homotrimeric protein consisting of 695 amino acid residues in each monomer. Although, the maximum activity of the enzyme was determined at pH 6.5 and 50°C, 60% of the maximum activity of the enzyme was determined at 25°C and 15% of the maximum activity was detected at 0°C. Conclusion The properties of Arthrobacter sp. 32cβ-D-galactosidase suggest that this enzyme could be useful for low-cost, industrial conversion of lactose into galactose and glucose in milk products and could be an interesting alternative for the production of ethanol from lactose-based feedstock.

  13. Cloning, Purification and Characterization of Acetyl Xylane Esterase from Anoxybacillus flavithermus DSM 2641(T) with Activity on Low Molecular-Weight Acetates.

    Science.gov (United States)

    Eminoğlu, Ayşenur; Ülker, Serdar; Sandallı, Cemal

    2015-08-01

    Family 4 carbohydrate esterases (CE-4) have deacetylate different forms of acetylated poly/oligosaccharides in nature. This family is recognized with a specific polysaccharide deacetylase domain assigned as NodB homology domain in their secondary structure. Most family 4 carbohydrate esterases have been structurally and biochemically characterized. However, this is the first study about the enzymological function of pdaB-like CE4s from thermophilic bacterium Anoxybacillus flavithermus DSM 2641(T). A. flavithermus WK1 genome harbors five putative CE4 family genes. One of them is 762 bp long and encodes a protein of 253 amino acids in length and it was used as reference sequence in this study. It was described as acetyl xylane esterase (AXE) in genome project and this AfAXE gene was amplified without signal sequence and cloned. The recombinant protein was expressed in E. coli BL21 (DE3), purified by nickel affinity chromatography and its purity was visualized on SDS-PAGE. The activity of the recombinant enzyme was shown by zymogram analysis with α-naphtyl acetate as a substrate. The enzyme was characterized spectrophotometrically using chromogenic p-nitrophenyl acetate. Optimum temperature and pH were determined as 50 °C and 7.5, respectively. Km and Vmax were determined as 0.43 mM and 3333.33 U/mg, respectively under optimum conditions. To our knowledge this is the first enzymological characterization of a pdaB-like family 4 carbohydrate esterase from the members of Anoxybacillus genus.

  14. Expression, purification, crystallization and preliminary X-ray crystallographic studies of a cold-adapted aspartate carbamoyltransferase from Moritella profunda

    Energy Technology Data Exchange (ETDEWEB)

    De Vos, Dirk; Hulpiau, Paco; Vergauwen, Bjorn; Savvides, Savvas N.; Van Beeumen, Jozef, E-mail: jozef.vanbeeumen@ugent.be [Laboratorium voor Eiwitbiochemie en Eiwitengineering, Universiteit Gent, K. L. Ledeganckstraat 35, Ghent (Belgium)

    2005-03-01

    Crystals of the aspartate carbamoyltransferase of the psychrophile M. profunda diffract X-rays to 2.85 Å. Three catalytic and three regulatory subunits are predicted per asymmetric unit. Aspartate carbamoyltransferase (ATCase) catalyzes the carbamoylation of the α-amino group of l-aspartate by carbamoyl phosphate (CP) to yield N-carbamoyl-l-aspartate and orthophosphate in the first step of de novo pyrimidine biosynthesis. Apart from its key role in nucleotide metabolism, the enzyme is generally regarded as a model system in the study of proteins exhibiting allosteric behaviour. Here, the successful preparation, crystallization and diffraction data collection of the ATCase from the psychrophilic bacterium Moritella profunda are reported. To date, there is no structural representative of a cold-adapted ATCase. The structure of M. profunda ATCase is thus expected to provide important insights into the molecular basis of allosteric activity at low temperatures. Furthermore, through comparisons with the recently reported structure of an extremely thermostable ATCase from Sulfolobus acidocaldarius, it is hoped to contribute to general principles governing protein adaptation to extreme environments. A complete native data to 2.85 Å resolution showed that the crystal belongs to space group P3{sub 2}21, with unit-cell parameters a = 129.25, b = 129.25, c = 207.23 Å, α = β = 90, γ = 120°, and that it contains three catalytic and three regulatory subunits per asymmetric unit. The three-dimensional structure of the Escherichia coli ATCase was sufficient to solve the structure of the M. profunda ATCase via the molecular-replacement method and to obtain electron density of good quality.

  15. Purification, characterization, primary structure, crystallization and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae ATCC 14544.

    Science.gov (United States)

    Kitadokoro, K; Tsuzuki, H; Nakamura, E; Sato, T; Teraoka, H

    1994-02-15

    A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase-2, was purified from the culture filtrate by S-Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDS/PAGE. When synthetic peptides were used as substrates, SFase-2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenylalaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase. The matured protein sequence of SFase-2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. SFase-2, consisting of 191 amino acids, is a novel proteinase. It showed 76% similarity in the amino acid sequence with Streptomyces griseus proteinase A [Johnson P. and Smillie L. B. (1974) FEBS Lett. 47, 1-6]. For insight into the three-dimensional structure of SFase-2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 6.92 nm, b = 7.28 nm, c = 2.99 nm; one molecule was present in the asymmetric unit.

  16. Purification, crystallization and X-ray crystallographic studies of a Bacillus cereus MepR-like transcription factor, BC0657.

    Science.gov (United States)

    Cho, Min Uk; Kim, Meong Il; Hong, Minsun

    2015-06-01

    Transcription factors of the MarR family respond to internal and external changes and regulate a variety of biological functions through ligand association with microorganisms. MepR belongs to the MarR family, and its mutations are associated with the development of multidrug resistance in Staphylococcus aureus, which has caused a growing health problem. In this study, a Bacillus cereus MepR-like transcription regulator, BC0657, was crystallized. The BC0657 crystals diffracted to 2.05 Å resolution and belonged to either space group P6(2)22 or P6(4)22, with unit-cell parameters a = 110.57, b = 110.57, c = 67.29 Å. There was one molecule per asymmetric unit. Future comparative structural studies on BC0657 would extend knowledge of ligand-induced transcriptional regulatory mechanisms in the MarR family and would make a significant contribution to the design of antibiotic drugs against multidrug-resistant bacteria.

  17. Cloning, periplasmic expression, purification and structural characterization of human ribosomal protein L10; Clonagem, expressao, purificacao e caracterizacao estrutural da proteina ribossomal L10 humana recombinante

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, Larissa Miranda

    2009-07-01

    The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm's tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribosomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813{sub Q}M at 25 degree C or 30 degree C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by {beta}- sheet feature. (author)

  18. 绵羊ISG15基因的克隆表达与纯化%The Cloning,Expression and Purification of Sheep ISG15 Gene

    Institute of Scientific and Technical Information of China (English)

    崔茹鹏; 沈文; 鲁海富; 孙延鸣

    2013-01-01

    To clone and express sheep ISG15,and purify its expression product,the gene of ISG15 was amplified by RT-PCR from sheep peripheral blood mononuclear cells (PBMC). The PCR product was inserted into the pMD18-T vector. The SIG15 cDNA confirmed by sequencing was inserted into expression vector pET-28a ( + ) and expressed in E. coli BL21 (DE3) strain. The ISG15 protein expression was induced by IPTG and assayed by SDS-PAGE and coomassie blue stain. The recombinant protein was purified by the method of Ni2+-NTA affinity chromatography. The result showed DNA sequencing confirmed that ISG15 gene length was 541bp,encoding 157 amino acids. The recombinant plasmid pET28a-ISG15 was transformed into E. coli BL21 (DE3) strain for expression. An expected 25 ku protein of ISG15 was found mainly in the induced host strains by SDS-PAGE and coomassie blue stain. The expressed protein was purified by the method of Ni2+ -NTA affinity chromatography. This study lays a foundation for further study of sheep ISG15.%为了克隆并表达绵羊ISG15,且对其表达产物进行纯化,首先从绵羊外周血单核细胞(PBMC)中提取总RNA,通过逆转录聚合酶链式反应(RT-PCR),扩增出ISG15的基因;再通过基因克隆技术,构建ISG15在pET28a中的重组表达质粒pET28a-ISG15,在大肠杆菌BL21 (DE3)用IPTG进行诱导表达,得到ISG15的原核表达蛋白;最后采用Ni2+-NTA亲和层析方法纯化目的蛋白.结果显示:克隆的绵羊ISG15基因序列长度为541 bp,编码157个氨基酸.重组表达产物通过SDS-PAGE可检测到相对分子质量为25 ku的重组蛋白,且表达的目的蛋白能被Ni2+-NTA亲和层析方法所纯化.该研究为后续深入研究绵羊ISG15奠定基础.

  19. Purification of mammalian DNA repair protein XRCC1

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-11-01

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

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

    Science.gov (United States)

    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.

  1. RNA Crystallization

    Science.gov (United States)

    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.

  2. What is Cloning?

    Science.gov (United States)

    Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...

  3. Cloning,Expression, Purification and Immunogenicity of House Dust Mite Allergen Der f5%粉尘螨Der f5克隆表达、纯化和免疫原性鉴定

    Institute of Scientific and Technical Information of China (English)

    袁小惠; 高安健; 邬玉兰; 刘志刚

    2013-01-01

    Objective To clone and express the antigen Der f5 from the dust mite dermatopha-goides farinae ,and to identify its immunogenicity. Methods A pair of primers were designed according to the known sequences of Der f 5 gene .Total RN A was extracted from dust mites and Der f5 gene fragments were amplified by RT-PCR .The PCR product was cloned into pMD18-T vector and transferred into E .coli Top10 .The clones were screened and identified by PCR and restriction enzyme digestion .The target gene obtained from the recombinant plasmid by digestion with Bam H Ⅰ and Hind Ⅲ was connected to the prokaryotic expression vector pET-32a.The expressed recombinant plasmid containing Der f5 gene was constructed by cloning target gene into pET-32a and transferred into E .coli Top10 .The recombinant plasmid was transfected into E .coli strain BL21 (DE3 ) and the transfected cells were grown in the presence of inducer IPTG .The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting and was purified by immo-bilized metal ion affinity chromatography. Results The two recombinant plasmids ,pMD18-T-Der f5 and pET32a-Der f5,were constructed successfully .SDS-PAGE showed a correct molecular weight of the recombinant Der f 5 protein .After purification by affinity chromatography ,the protein showed only one strip on SDS-PAGE gel and appropriate ability to bind to IgE in sera of allergic patients .Conclusion The Der f5 gene has been cloned,expressed and purified and the recombinant Der f5 protein shows appropriate IgE-combined ability . The study provides a basis for specific diagnosis and treatment of dust mite-induced allergic disease.%目的 克隆表达粉尘螨第5组变应原(dermatophagoides farinae,Der f5)基因,并鉴定纯化蛋白免疫原性.方法 根据Der f5 基因已知序列,设计出相应的引物,提取粉尘螨总RNA,采用RT-PCR 方法扩增出Der f5 基因片段,PCR 产物克隆入pMD18-T 载体,转化大肠埃希菌 Top10,经PCR和酶切鉴定

  4. A nifS-like gene, csdB, encodes an Escherichia coli counterpart of mammalian selenocysteine lyase. Gene cloning, purification, characterization and preliminary x-ray crystallographic studies.

    Science.gov (United States)

    Mihara, H; Maeda, M; Fujii, T; Kurihara, T; Hata, Y; Esaki, N

    1999-05-21

    Selenocysteine lyase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of L-selenocysteine to L-alanine and elemental selenium. An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii. In this study, the csdB gene was cloned and expressed in E. coli cells. The gene product was a homodimer with the subunit Mr of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from L-selenocysteine, L-cysteine sulfinic acid, and L-cysteine, respectively, to yield L-alanine; the reactivity of the substrates decreased in this order. Although the enzyme was not specific for L-selenocysteine, the high specific activity for L-selenocysteine (5.5 units/mg compared with 0.019 units/mg for L-cysteine) supports the view that the enzyme can be regarded as an E. coli counterpart of mammalian selenocysteine lyase. We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method. The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 A and c = 137.0 A. Consideration of the Matthews parameter Vm (3.19 A3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit. A native diffraction dataset up to 2.8 A resolution was collected. This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family.

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

    DEFF Research Database (Denmark)

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

    1993-01-01

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

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

    DEFF Research Database (Denmark)

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

    1993-01-01

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

  7. Crystallization and preliminary crystallographic studies of a cysteine protease inhibitor from the human nematode parasite Ascaris lumbricoides.

    Science.gov (United States)

    Liu, Sanling; Dong, Jianmei; Mei, Guoqiang; Liu, Guiyun; Xu, Wei; Su, Zhong; Liu, Jinsong

    2011-02-01

    The cysteine protease inhibitor from Ascaris lumbricoides, a roundworm that lives in the human intestine, may be involved in the suppression of human immune responses. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of the cysteine protease inhibitor from A. lumbricoides are reported. The rod-shaped crystal belonged to space group C2, with unit-cell parameters a = 99.40, b = 37.52, c = 62.92 Å, β = 118.26°. The crystal diffracted to 2.1 Å resolution and contained two molecules in the asymmetric unit.

  8. Cloning, expression, purification and antigenicity analysis of AhpC in Campylobacter jejuni%空肠弯曲菌AhpC蛋白的克隆表达及抗原性分析

    Institute of Scientific and Technical Information of China (English)

    曹芳芳; 孟凡亮; 乔博; 张茂俊; 张建中

    2011-01-01

    目的 构建空肠弯曲菌烷基过氧化氢还原酶(ah pC)基因的原核表达系统,克隆表达AhpC蛋白,用Western blot以及ELISA方法检测表达蛋白的抗原性,为筛选空肠弯曲菌感染后特异性抗体检测试剂的制备奠定基础.方法 用PCR方法从中国分离菌株ICDCCJ07001的染色体DNA中扩增出ah pC基因,将目的基因插入表达载体pGEX-4T-1后转化入大肠杆菌E.coliBL21( DE3),IPTG诱导重组质粒pG EX-ah pC的表达.SDS-PAGE分析表达产物,采用GST标签亲和层析柱纯化表达蛋白.应用兔免疫血清、临床感染者血清以及健康无感染者血清,利用Western blot以及ELISA的方法分析AhpC蛋白的抗原性并初步评价其在ELISA检测方法中的应用.结果 PCR扩增及双酶切鉴定证实重组质粒构建成功.重组表达蛋白经飞行质谱鉴定为空肠弯曲菌AhpC蛋白,Western blot及ELISA检测结果显示AhpC具有特异抗原性.结论 成功构建了ah pC重组表达载体pGEX-4T-1/ahpC,并在大肠杆菌中高效表达.空肠弯曲菌AhpC蛋白具有抗原性,可以作为空肠弯曲菌感染后血清抗体检测的特异抗原.%To clone and express the AhpC protein of Campylobacter jejuni in E. Coli and characterize its antigenicity and specificity. The ah pc gene was amplified by PCR from C. Jejeuni strain ICDCCJ07001 < and cloned into pGEX-4T-l. The recombinant PGEX-4T-l-ah pC was transformed to E. Coli BL21(DE3) for protein expression. The expression product was analyzed and identified by SDS-PAGE and MALD1 TOF-TOF 4700 Proteomics Analyzer. The expressed protein was identified as Campylobacter jejuni AhpC protein by Proteomics Analyzer. Both Western blot and ELISA showed that AhpC had specific antigenicity for Campylobacter jejuni. The expression and purification of recombinant AhpC antigen with good antigenicity and specificity will facilitate the research and clinical application in the diagnosis of C. Jejuni infection.

  9. Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Delta(4)-(5 alpha)-dehydrogenase from Rhodococcus jostii RHA1

    NARCIS (Netherlands)

    van Oosterwijk, Niels; Knol, Jan; Dijkhuizen, Lubbert; van der Geize, Robert; Dijkstra, Bauke

    2011-01-01

    3-Ketosteroid dehydrogenases are flavoproteins which play key roles in steroid ring degradation. The enzymes are abundantly present in actinobacteria, including the catabolic powerhouse Rhodococcus jostii and the pathogenic species R. equi and Mycobacterium tuberculosis. The gene for 3-ketosteroid

  10. Hamiltonian purification

    Energy Technology Data Exchange (ETDEWEB)

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

    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. Quantum cloning

    OpenAIRE

    Scarani, Valerio; Iblisdir, Sofyan; Gisin, Nicolas; Acin, Antonio

    2005-01-01

    The impossibility of perfectly copying (or cloning) an arbitrary quantum state is one of the basic rules governing the physics of quantum systems. The processes that perform the optimal approximate cloning have been found in many cases. These "quantum cloning machines" are important tools for studying a wide variety of tasks, e.g. state estimation and eavesdropping on quantum cryptography. This paper provides a comprehensive review of quantum cloning machines (both for discrete-dimensional an...

  12. Cloning,expression and purification of dust mite ferritin and its molecular characteristics%粉尘螨铁蛋白的克隆、表达及蛋白特征分析

    Institute of Scientific and Technical Information of China (English)

    林建立; 詹政科; 刘玉琳; 范小琴; 耿晓瑞; 刘晓宇

    2015-01-01

    expressed recombinant plasmid containing ferritin gene was constructed by cloning target gene into pET‐32a and transferred into E .coli Bl21 (DE3) .The ex‐pressed recombinant protein was analyzed by SDS‐PAGE ,and was purified by immobilized metal ion affinity chromatography (IMAC) .The ferritin expressed by dust mite was analyzed by the method of bioinformatics .The recombinant plasmid pET32a‐ferritin was constructed .SDS‐PAGE showed a correct molecular weight of the recombinant ferritin protein .After purification by affinity chromatography ,the protein showed only one strip on SDS‐PAGE gel .SDS‐PAGE showed a band at 20 kD .Dust mite ferritin contains 8 serine kinase ,7 threonine kinase ,7 tyrosine kinase ,and 0 histidine kinase phosphorylation sites .Hy‐drophilic region is larger than the hydrophobic region and it is an unstable protein .In conclusion ,the ferritin gene has been cloned and expressed .The purified ferritin has high purity . The study provides a basis for further study of composition and physicochemical properties of house dust mite allergen .

  13. Purification and Characterization of Tryptophan Hydroxylase

    DEFF Research Database (Denmark)

    Haahr, Lærke Tvedebrink

    This thesis deals with the purification and characterization of the iron-containing enzyme tryptophan hydroxylase (TPH). TPH exists in two isoforms, called TPH1 and TPH2. Each isoform consists of threestructural distinct domains: the regulatory, the catalytic and the tetramerization domain. TPH...... of this project was to developpurification methods for full-length TPH1 and TPH2 as well as to characterize purified TPH variants. A successful purification method for full-length human TPH1 (hTPH1) was developed, which resulted in pure, active and stable protein. The method includes affinity-purification using....... The crystallization procedure for the catalytic domain of gallus gallus TPH1 (cgTPH1) was optimized to faster crystal growth by addition of tryptophan and incubation at room temperature. Crystals without imidazole in the crystallization conditions could be obtained. The solved structures were however of poor quality...

  14. A modified version of the digestion-ligation cloning method for more efficient molecular cloning.

    Science.gov (United States)

    Gao, Song; Li, Yanling; Zhang, Jiannan; Chen, Hongman; Ren, Daming; Zhang, Lijun; An, Yingfeng

    2014-05-15

    Here we describe a modified version of the digestion-ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and blunt-end cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.

  15. Polonium purification

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Academic Cloning.

    Science.gov (United States)

    Sikula, John P.; Sikula, Andrew F.

    1980-01-01

    The authors define "cloning" as an integral feature of all educational systems, citing teaching practices which reward students for closely reproducing the teacher's thoughts and/or behaviors and administrative systems which tend to promote like-minded subordinates. They insist, however, that "academic cloning" is not a totally…

  17. Purification, crystallization and preliminary X-ray analysis of bifunctional isocitrate dehydrogenase kinase/phosphatase in complex with its substrate, isocitrate dehydrogenase, from Escherichia coli

    OpenAIRE

    2009-01-01

    The protein complex of bifunctional isocitrate dehydrogenase kinase/phosphatase with its substrate, isocitrate dehydrogenase, has been crystallized for structural analysis. A complete data set was collected from the complex crystal and processed to 2.9 Å resolution.

  18. The expression, purification, crystallization and preliminary X-ray analysis of a subcomplex of the peripheral stalk of ATP synthase from bovine mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Silvester, Jocelyn A.; Kane Dickson, Veronica; Runswick, Michael J. [The Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY (United Kingdom); Leslie, Andrew G. W. [The Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH (United Kingdom); Walker, John E., E-mail: walker@mrc-dunn.cam.ac.uk [The Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY (United Kingdom)

    2006-06-01

    A recombinant subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been crystallized and a native data set has been collected to 2.8 Å resolution. A subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been expressed to high levels in a soluble form in Escherichia coli. The subcomplex consists of residues 79–184 of subunit b, residues 1–124 of subunit d and the entire F{sub 6} subunit (76 residues). It has been purified and crystallized by vapour diffusion. The morphology and diffraction properties of the crystals of the subcomplex were improved by the presence of thioxane or 4-methylpyridine in the crystallization liquor. With a synchrotron-radiation source, these crystals diffracted to 2.8 Å resolution. They belong to the monoclinic space group P2{sub 1}.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rohman, Ali [Department of Chemistry, Faculty of Mathematics and Natural Sciences, Airlangga University, Kampus C Unair, Jl. Mulyorejo, Surabaya 60115 (Indonesia); Oosterwijk, Niels van [Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen (Netherlands); Kralj, Slavko; Dijkhuizen, Lubbert [Laboratory of Microbial Physiology, University of Groningen, Kerklaan 30, 9750 NN Haren (Netherlands); Dijkstra, Bauke W. [Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen (Netherlands); Puspaningsih, Ni Nyoman Tri, E-mail: nyomantri@unair.ac.id [Department of Chemistry, Faculty of Mathematics and Natural Sciences, Airlangga University, Kampus C Unair, Jl. Mulyorejo, Surabaya 60115 (Indonesia)

    2007-11-01

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

  20. Investigation on Purification of Dimethyl Terephthalate Using Falling Film Crystallization%降膜结晶法精制对苯二甲酸二甲酯的研究

    Institute of Scientific and Technical Information of China (English)

    孙绪江; 张军; 齐彦伟; 符强

    2001-01-01

    对苯二甲酸二甲酯(DMT)是生产聚酯的基本原料,由于精制工艺流程长,导致DMT成本高,降低了竞争力。本文将降膜结晶技术用于DMT的分离精制,得到了高纯度的产品,讨论了各种操作条件对结晶速度和产品纯度的影响,确定了最优操作条件。%Dimethyl terephthalate(DMT)is the main raw material for producingpolyester fiber.Because its purification process is long,so the cost is very high.In this paper,the falling film crystallization is used to separate and purify DMT.The product of high purity is obtained.The influence of various operating conditions on the crystallization rate and product purity have been investigated.The optimum operating condition is given.

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

    DEFF Research Database (Denmark)

    Wilkens, Casper; Poulsen, Jens-Christian Navarro; Ramløv, Hans

    2014-01-01

    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...

  2. A second gene for acyl-(acyl-carrier-protein): glycerol-3-phosphate acyltransferase in squash, Cucurbita moschata cv. Shirogikuza(*), codes for an oleate-selective isozyme: molecular cloning and protein purification studies.

    Science.gov (United States)

    Nishida, I; Sugiura, M; Enju, A; Nakamura, M

    2000-12-01

    A new isogene for acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) in squash has been cloned and the gene product was identified as oleate-selective GPAT. Using PCR primers that could hybridise with exons for a previously cloned squash GPAT, we obtained two PCR products of different size: one coded for a previously cloned squash GPAT corresponding to non-selective isoforms AT2 and AT3, and the other for a new isozyme, probably the oleate-selective isoform AT1. Full-length amino acid sequences of respective isozymes were deduced from the nucleotide sequences of genomic genes and cDNAs, which were cloned by a series of PCR-based methods. Thus, we designated the new gene CmATS1;1 and the other one CmATS1;2. Genome blot analysis revealed that the squash genome contained the two isogenes at non-allelic loci. AT1-active fractions were partially purified, and three polypeptide bands were identified as being AT1 polypeptides, which exhibited relative molecular masses of 39.5-40.5 kDa, pI values of 6.75-7.15, and oleate selectivity over palmitate. Partial amino-terminal sequences obtained from two of these bands verified that the new isogene codes for AT1 polypeptides.

  3. Purification and separation of durene by static melt crystallization☆

    Institute of Scientific and Technical Information of China (English)

    Shan Cong; Ying Liu; Hong Li; Xingang Li; Lvhong Zhang; Lei Wang

    2015-01-01

    The purification and separation of durene from the mixture containing durene isomers were studied. Since the boiling points of tetramethyl benzene isomers are very close but their melting points are of great differences, stat-ic melt crystallization was applied to separate and purify durene from its isomers. Crystallization experiments were carried out under various operating conditions. The effects of cooling rate, crystallization temperature, sweating temperature and sweating time on the yield and purity of crystal were investigated. Orthogonal exper-imental design method was adopted to analyze the factors that may affect the yield of durene. Under the optimal crystal ization conditions, the purity of durene could reach as high as 99.06%with the yield of 75.3%through one crystal ization process. By fitting purification data based on sweating time in isothermal operations, the purifica-tion rate coefficient was obtained.

  4. Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB

    Energy Technology Data Exchange (ETDEWEB)

    Pyburn, Tasia M.; Yankovskaya, Victoria; Bensing, Barbara A.; Cecchini, Gary; Sullam, Paul M.; Iverson, T.M. (VA); (Vanderbilt); (UCSF)

    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.

  5. Purification, Crystallization and Preliminary X-ray Characterization of Prunin-1, a Major Component of the Almond (Prunus dulcis) Allergen Amandin

    Energy Technology Data Exchange (ETDEWEB)

    Albillos, Silvia M.; Jin, Tengchuan; Howard, Andrew; Zhang, Yuzhu; Kothary, Mahendra H.; Fu, Tong-Jen (IIT); (US-FDA)

    2008-08-04

    The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond (Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 {angstrom} and belong to the tetragonal space group P4{sub 1}22, with unit cell parameters of a = b = 150.912 {angstrom}, c = 165.248 {angstrom}. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

  6. Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum {beta}-lactamase conferring severe antibiotic resistance

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J. H., E-mail: msgjhlee@mju.ac.kr; Sohn, S. G., E-mail: sgsohn@mju.ac.kr; Jung, H. I., E-mail: jhinumber1@hanmail.net; An, Y. J., E-mail: anyj0120@hanmail.net; Lee, S. H., E-mail: sangheelee@mju.ac.kr [Myongji University, Drug Resistance Proteomics Laboratory, Department of Biological Sciences (Korea, Republic of)

    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%.

  7. Purification, crystallization and preliminary X-ray diffraction analysis of the CBS-domain pair from the Methanococcus jannaschii protein MJ0100

    Science.gov (United States)

    Lucas, María; Kortazar, Danel; Astigarraga, Egoitz; Fernández, José A.; Mato, Jose M.; Martínez-Chantar, María Luz; Martínez-Cruz, Luis Alfonso

    2008-01-01

    CBS domains are small protein motifs consisting of a three-stranded β-sheet and two α-helices that are present in proteins of all kingdoms of life and in proteins with completely different functions. Several genetic diseases in humans have been associated with mutations in their sequence, which has made them promising targets for rational drug design. The C-terminal domain of the Methanococcus jannaschii protein MJ0100 includes a CBS-domain pair and has been overexpressed, purified and crystallized. Crystals of selenomethionine-substituted (SeMet) protein were also grown. The space group of both the native and SeMet crystals was determined to be orthorhombic P212121, with unit-cell parameters a = 80.9, b = 119.5, c = 173.3 Å. Preliminary analysis of the X-ray data indicated that there were eight molecules per asymmetric unit in both cases. PMID:18931440

  8. Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum β-lactamase conferring severe antibiotic resistance

    Science.gov (United States)

    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%.

  9. Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Rengachari, Srinivasan; Aschauer, Philipp; Sturm, Christian; Oberer, Monika, E-mail: m.oberer@uni-graz.at [University of Graz, Humboldtstrasse 50/3, 8010 Graz (Austria)

    2015-01-28

    A soluble variant of the monoglyceride lipase Yju3p was successfully expressed, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space group P2{sub 1}2{sub 1}2{sub 1}), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

  10. Overproduction, purification, crystallization and preliminary X-ray analysis of the peroxiredoxin domain of a larger natural hybrid protein from Thermotoga maritima

    Energy Technology Data Exchange (ETDEWEB)

    Barbey, Carole, E-mail: carole.barbey@smbh.univ-paris13.fr [Laboratoire de Biophysique Moléculaire, Cellulaire et Tissulaire, UMR 7033, Université Paris 13, UFR SMBH, 74 Rue Marcel Cachin, 93017 Bobigny CEDEX (France); Rouhier, Nicolas [Unité Mixte de Recherches 1136 INRA UHP (Interaction Arbres Microorganismes), IFR 110, Nancy Université BP 239, 54506 Vandoeuvre-lès-Nancy CEDEX (France); Haouz, Ahmed [Plate-forme de Cristallogenèse et Diffraction des Rayons X, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris (France); Navaza, Alda [Laboratoire de Biophysique Moléculaire, Cellulaire et Tissulaire, UMR 7033, Université Paris 13, UFR SMBH, 74 Rue Marcel Cachin, 93017 Bobigny CEDEX (France); Jacquot, Jean-Pierre [Unité Mixte de Recherches 1136 INRA UHP (Interaction Arbres Microorganismes), IFR 110, Nancy Université BP 239, 54506 Vandoeuvre-lès-Nancy CEDEX (France); Laboratoire de Biophysique Moléculaire, Cellulaire et Tissulaire, UMR 7033, Université Paris 13, UFR SMBH, 74 Rue Marcel Cachin, 93017 Bobigny CEDEX (France)

    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.

  11. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Bækgaard, Lone

    2010-01-01

    -bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space......Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example...

  12. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Palmgren, Michael Broberg

    2010-01-01

    -bound calmodulin (Ca2+-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space......Plasma-membrane Ca2+-ATPases (PMCAs) are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance, with different isoforms being essential, for example...

  13. Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

    Science.gov (United States)

    Leong, L. E.-C.; Walker, P. A.; Porter, A. G.

    1992-08-01

    The protease (3C pro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3C pro has been achieved by fusing the protein to the car☐y-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3C pro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3C pro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3C pro. Sufficient protease 3C pro has been purified for initial attempts at crystallization.

  14. Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chunmao [State Key Laboratory of Pathogen and Biosecurity, Beijng Institute of Microbiology and Infectious Disease, No. 20 Dongda Street, Fengtai District, Beijing 100071 (China); Yu, You [Key Laboratory for Protein Sciences of Ministry of Education, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing (China); Yang, Maojun, E-mail: maojunyang@tsinghua.edu.cn [Key Laboratory for Protein Sciences of Ministry of Education, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing (China); Jiang, Yongqiang, E-mail: jiangyq@bmi.ac.cn [State Key Laboratory of Pathogen and Biosecurity, Beijng Institute of Microbiology and Infectious Disease, No. 20 Dongda Street, Fengtai District, Beijing 100071 (China)

    2015-10-23

    Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. We speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.

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

    National Research Council Canada - National Science Library

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    .... The crystal belongs to the orthorhombic space group P 2 1 2 1 2 1 , with unit‐cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self...

  16. PURIFICATION, CRYSTALLIZATION AND PRELIMINARY-X-RAY ANALYSIS OF PENICILLIN-BINDING PROTEIN-4 FROM ESCHERICHIA-COLI, A PROTEIN RELATED TO CLASS-A BETA-LACTAMASES

    NARCIS (Netherlands)

    THUNNISSEN, MMGM; FUSETTI, F; DEBOER, B; DIJKSTRA, BW

    1995-01-01

    Crystals of the penicillin binding protein 4 (PBP4) from Escherichia coli have been obtained at 37 degrees C from liquid to liquid diffusion experiments in capillaries. PBP4 was dissolved in a 1.0 M ammonium sulphate solution, buffered at pH 7.2, to a concentration of 5 mg/ml, and was layered on top

  17. Molecular cloning.

    Science.gov (United States)

    Lessard, Juliane C

    2013-01-01

    This protocol describes the basic steps involved in conventional plasmid-based cloning. The goals are to insert a DNA fragment of interest into a receiving vector plasmid, transform the plasmid into E. coli, recover the plasmid DNA, and check for correct insertion events.

  18. Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties.

    Science.gov (United States)

    Don Paul, Craig; Traore, Daouda A K; Byres, Emma; Rossjohn, Jamie; Devenish, Rodney J; Kiss, Csaba; Bradbury, Andrew; Wilce, Matthew C J; Prescott, Mark

    2011-10-01

    Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, β = 89.92, γ = 104.03°. The Matthews coefficient (V(M) = 2.26 Å(3) Da(-1)) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.

  19. Overproduction, purification and crystallization of a chondroitin sulfate A-binding DBL domain from a Plasmodium falciparum var2csa-encoded PfEMP1 protein

    Energy Technology Data Exchange (ETDEWEB)

    Higgins, Matthew K., E-mail: mkh20@cam.ac.uk [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom)

    2008-03-01

    A chondroitin sulfate A-binding DBL important in placental malaria has been overproduced, purified and crystallized. Diffraction data were collected to 1.9 Å resolution. The PfEMP1 proteins of the malaria parasite Plasmodium falciparum are inserted into the membrane of infected red blood cells, where they mediate adhesion to a variety of human receptors. The DBL domains of the var2csa-encoded PfEMP1 protein play a critical role in malaria of pregnancy, tethering infected cells to the surface of the placenta through interactions with the glycosaminoglycan carbohydrate chondroitin sulfate A (CSA). A CSA-binding DBL domain has been overproduced in a bacterial expression system, purified and crystallized. Native data sets extending to 1.9 Å resolution have been collected and phasing is under way.

  20. Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490963–1138 domain of TamB from Escherichia coli

    Science.gov (United States)

    Josts, Inokentijs; Grinter, Rhys; Kelly, Sharon M.; Mosbahi, Khedidja; Roszak, Aleksander; Cogdell, Richard; Smith, Brian O.; Byron, Olwyn; Walker, Daniel

    2014-01-01

    TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963–1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Å resolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future. PMID:25195908

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

    Science.gov (United States)

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

    2014-05-01

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

  2. Purification, crystal structure and antimicrobial activity of phenazine-1-carboxamide produced by a growth-promoting biocontrol bacterium, Pseudomonas aeruginosa MML2212.

    Science.gov (United States)

    Shanmugaiah, V; Mathivanan, N; Varghese, B

    2010-02-01

    To purify and characterize an antimicrobial compound produced by a biocontrol bacterium, Pseudomonas aeruginosa MML2212, and evaluate its activity against rice pathogens, Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Pseudomonas aeruginosa strain MML2212 isolated from the rice rhizosphere with wide-spectrum antimicrobial activity was cultured in Kings'B broth using a fermentor for 36 h. The extracellular metabolites were isolated from the fermented broth using ethyl acetate extraction and purified by two-step silica-gel column chromatography. Three fractions were separated, of which a major compound was obtained in pure state as yellow needles. It was crystallized after dissolving with chloroform followed by slow evaporation. It is odourless with a melting point of 220-222 degrees C. It was soluble in most of the organic solvents and poorly soluble in water. The molecular mass of purified compound was estimated as 223.3 by mass spectral analysis. Further, it was characterized by IR, (1)H and (13)C NMR spectral analyses. The crystal structure of the compound was elucidated for the first time by X-ray diffraction study and deposited in the Cambridge Crystallographic Data Centre (http://www.ccde.com.ac.uk) with the accession no. CCDC 617344. The crystal compound was undoubtedly identified as phenazine-1-carboxamide (PCN) with the empirical formula of C(13)H(9)N(3)O. As this is the first report on the crystal structure of PCN, it provides additional information to the structural chemistry. Furthermore, the present study reports the antimicrobial activity of purified PCN on major rice pathogens, R. solani and X. oryzae pv. oryzae. Therefore, the PCN can be developed as an ideal agrochemical candidate for the control of both sheath blight and bacterial leaf blight diseases of rice.

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

    Science.gov (United States)

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

    2012-03-01

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

  4. Human Cloning

    Science.gov (United States)

    2006-07-20

    genes , for example, has led to new treatments developed by the biotechnology industry for diseases such as diabetes and hemophilia . In the context of...stem cells should be permitted because of the potential for developing new therapies and advancing biomedical knowledge. On May 24, 2005, the House...to describe many different processes that involve making copies of biological material, such as a gene , a cell, a plant or an animal. The cloning of

  5. Purification, crystallization and preliminary X-ray diffraction analysis of inner membrane complex (IMC) subcompartment protein 1 (ISP1) from Toxoplasma gondii.

    Science.gov (United States)

    Tonkin, Michelle L; Brown, Shannon; Beck, Josh R; Bradley, Peter J; Boulanger, Martin J

    2012-07-01

    The protozoan parasites of the Apicomplexa phylum are devastating global pathogens. Their success is largely due to phylum-specific proteins found in specialized organelles and cellular structures. The inner membrane complex (IMC) is a unique apicomplexan structure that is essential for motility, invasion and replication. The IMC subcompartment proteins (ISP) have recently been identified in Toxoplasma gondii and shown to be critical for replication, although their specific mechanisms are unknown. Structural characterization of TgISP1 was pursued in order to identify the fold adopted by the ISPs and to generate detailed insight into how this family of proteins functions during replication. An N-terminally truncated form of TgISP1 was purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis. Two crystal forms of TgISP1 belonging to space groups P4(1)32 or P4(3)32 and P2(1)2(1)2(1) diffracted to 2.05 and 2.1 Å resolution, respectively.

  6. Expression, refolding, purification and crystallization of the sensory domain of the TlpC chemoreceptor from Helicobacter pylori for structural studies.

    Science.gov (United States)

    Liu, Yu Chih; Roujeinikova, Anna

    2015-03-01

    Helicobacter pylori infections are associated with gastritis, duodenal and gastric ulcers and gastric adenocarcinoma. Bacterial chemotaxis, mediated by four different chemoreceptors (also termed transducer-like proteins (Tlp)), plays an important role in initial colonization and development of disease. Chemoreceptor sensory domains of H. pylori share no significant sequence similarity with those of Escherichia coli or any other non-Epsilonproteobacteria. The structural basis of how chemical signals are recognized by chemoreceptors of H. pylori is poorly understood mainly due to the lack of a robust procedure to purify their sensory domains in a soluble form. This study reports a method for extraction of the periplasmic sensory domain of transducer-like protein C (TlpC) from inclusion bodies and refolding to yield 5mg pure crystallizable protein per 1l of bacterial culture. Purified protein was monomeric in solution by size-exclusion chromatography and folded according to the circular dichroism spectrum. Crystals have been grown by the hanging-drop vapor-diffusion method using PEG 4000 as a precipitating agent. The crystals belonged to space group C2, with unit-cell parameters a=189.3, b=103.2, c=61.8Å, β=98.3. A complete X-ray diffraction data set has been collected to 2.2 Å resolution using cryocooling conditions and synchrotron radiation. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains three monomers.

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

    Energy Technology Data Exchange (ETDEWEB)

    Logsdon, Naomi J.; Allen, Christopher E.; Rajashankar, Kanagalaghatta R.; Walter, Mark R. (Cornell); (UAB)

    2012-02-08

    Interleukin-20 (IL-20) is an IL-10-family cytokine that regulates innate and adaptive immunity in skin and other tissues. In addition to protecting the host from various external pathogens, dysregulated IL-20 signaling has been shown to contribute to the pathogenesis of human psoriasis. IL-20 signals through two cell-surface receptor heterodimers, IL-20R1-IL-20R2 and IL-22R1-IL-20R2. In this report, crystals of the IL-20-IL-20R1-IL-20R2 ternary complex have been grown from polyethylene glycol solutions. The crystals belonged to space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = 111, c = 135 {angstrom}, and diffracted X-rays to 3 {angstrom} resolution. The crystallographic asymmetric unit contains one IL-20-IL-20R1-IL-20R2 complex, corresponding to a solvent content of approximately 54%.

  8. Peptide recognition by two HLA-A2/Tax(11-19)-specific T cell clones in relationship to their MHC/peptide/TCR crystal structures

    NARCIS (Netherlands)

    Hausmann, S; Biddison, WE; Smith, KJ; Ding, YH; Garboczi, DN; Utz, U; Wiley, DC; Wucherpfennig, KW

    1999-01-01

    The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues

  9. Cloning, expression and purification of arginine kinase from Locusta migratoria manilensis and its allergic activity%东亚飞蝗主要过敏原精氨酸激酶基因的克隆表达、纯化及免疫原性鉴定

    Institute of Scientific and Technical Information of China (English)

    陈义昆; 邬玉兰; 李荔; 连国云; 刘志刚

    2013-01-01

    This investigation aimed to clone the Locusta migratoria manilensis arginine kinase (AK) gene, produce its recombinant protein and investigate the protein ' s allergenicity. The cDNA of AK was cloned, using specific primers, from the total RNA of L. m. manilensis. The cloned gene was inserted into pMD18-T vector and digested by EcoR I and Xho I. The cDNA was sequenced and subcloned into a pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli Rosetta by IPTG induction. The recombinant AK (rAK) was purified by metal ( Ni2+) chelating affinity chromatography. Its allergenicity was examined by Western blotting. The cloned cDNA ORF sequence contained 1 068 bp and encoded 355 amino acids. Its sequence homology with the published sequence (Accession no. DQ513322) was 98% at nucleotide level. The allergen rAK was highly expressed in E. coli as a soluble protein with a molecular weight of about Mr 40 000 under induction with IPTG and purified by a 6-His-tag purification system. Under both non-denaturalization and denaturalization conditions, the recombinant allergen was identified by its affinity to IgE antibodies from the cockroach-allergic patient sera by Western blotting. It is concluded that recombinant arginine kinase with proper allergenicity was successfully obtained.%本研究克隆了东亚飞蝗Locusta migratoria manilensis (Meyen)精氨酸激酶(arginine kinase,AK)基因全长,表达并纯化重组AK蛋白,研究重组蛋白的免疫反应性.东亚飞蝗AK基因开放阅读框全长为1 068 bp,编码355个氨基酸,与GenBank中已登录的东亚飞蝗AK(DQ513322)基因同源性为98%,重组质粒pET-28 a-AK在E.coli中获得高效表达,重组蛋白相对分子质量(Mr)约为40 000,主要以可溶性形式表达,经亲和层析获得重组蛋白.通过免疫印迹分析结果表明,重组AK蛋白可被过敏性患者血清识别,免疫原性良好.结果表明我们成功获得东亚飞蝗精氨酸激

  10. Cloning, expression, crystallization and preliminary X-ray studies of the ferredoxin-NAD(P)+ reductase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

    Science.gov (United States)

    Liauw, Pasqual; Mashiba, Tomohiro; Kopczak, Marta; Wiegand, Katrin; Muraki, Norifumi; Kubota, Hisako; Kawano, Yusuke; Ikeuchi, Masahiko; Hase, Toshiharu; Rögner, Matthias; Kurisu, Genji

    2012-09-01

    Ferredoxin-NADP(+) reductase (FNR) is a flavoenzyme that catalyses the reduction of NADP(+) in the final step of the photosynthetic electron-transport chain. FNR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TeFNR) contains an additional 9 kDa domain at its N-terminus relative to chloroplastic FNRs and is more thermostable than those from mesophilic cyanobacteria. With the aim of understanding the structural basis of the thermostability of TeFNR and assigning a structural role to the small additional domain, the gene encoding TeFNR with and without an additional domain was engineered for heterologous expression and the recombinant proteins were purified and crystallized. Crystals of TeFNR without the additional domain belonged to space group P2(1), with unit-cell parameters a = 55.05, b = 71.66, c = 89.73 Å, α = 90, β = 98.21, γ = 90°.

  11. Cloning,over-expression and purification of the recombinant peroxiredoxin protein of Toxoplasma gondii%刚地弓形虫Peroxiredoxin基因的克隆及其融合蛋白的高效表达和纯化

    Institute of Scientific and Technical Information of China (English)

    李雪艳; 刘转转; 付琳琳; 颜超; 杜文平; 汤仁仙; 郑葵阳; 刘宜升

    2012-01-01

    [目的]克隆刚地弓形虫Prx基因,用IPTG诱导表达Prx融合蛋白并进行纯化.[方法]用PCR扩增目的基因片段并克隆至pGEX-6P-1载体,构建pGEX-6p-1/TgPrx原核表达载体;IPTG诱导表达Prx融合蛋白,表达产物进行SDS-PAGE和Western blot分析,GST亲和层析纯化融合蛋白.[结果]从弓形虫RH株DNA中扩增Prx基因,成功构建了弓形虫重组质粒pGEX-6p-1/TgPrx,并在大肠埃希菌(E.coli)中得到高效表达,表达的融合蛋白分子质量为51ku,该蛋白可被鼠抗弓形虫血清特异性识别.[结论]原核表达具有生物学活性的弓形虫重组Prx蛋白(rTgPrx),该蛋白有望用于弓形虫病免疫学检测.%Objective To clone the Peroxiredoxin (Prx) gene of Toxoplasma gondiiand to generate a prokaryotic expression vector to obtain a fusion protein of Prx with a GST tag Methods Prx gene fragments amplified by PCR were cloned into the pGEX-6P-l vector to construct the prokaryotic expression vector pGEX-6p-l/TgPrx. Expression of the recombinant protein Prx (rTgPrx) was then induced by IPTG in E. coli BI.21. Expressed products were purified by affinity ehromatography and identified by SDS-PAGE, and Western blotting. Results The fusion protein rTgPrx with a molecular weight of 51 ku was efficiently expressed in E. coli BL21, and bound specifically to mouse polyclonal antibodies a-gainst Toxoplasma gondii. Conclusion A prokaryotic expression vector that expresses the recombinant protein Prx was successfully created and may provide the foundation for producing large quantities of rTgPrx to clinically diagnose human toxoplasmosis.

  12. Enabling structure-based drug design of Tyk2 through co-crystallization with a stabilizing aminoindazole inhibitor

    Directory of Open Access Journals (Sweden)

    Argiriadi Maria A

    2012-09-01

    Full Text Available Abstract Background Structure-based drug design (SBDD can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds. Results A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5–2.6 Å resolution. This structure revealed initial “hot-spot” regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010 J Mol Biol 400:413, our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods. Conclusions Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process

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

    OpenAIRE

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

    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/de...

  14. Expression, purification and crystallization of Human FCHo1 μHD domain%人源 FCHo1蛋白μHD 结构域的表达、纯化及晶体生长

    Institute of Scientific and Technical Information of China (English)

    李夫; 夏鹏飞; 戴雪辰; 石康; 熊鹰; 刘映乐

    2015-01-01

    人FCHo1蛋白(Fer/Cip4 homology domain only proteins 1)是网格蛋白介导的内吞途径中的成核剂,其碳端μHD结构域( AP2-μhomology domain)对于FCHo1在披网格蛋白小泡( clathrin-coated vesicle)形成过程中的正确定位非常重要。为揭示人FCHo1碳端μHD结构域的作用机制,制备可衍射的μHD结构域蛋白质晶体并解析其结构至关重要。通过构建pET15b原核表达载体,并转化大肠杆菌BL21(DE3),诱导表达。利用亲和层析、离子交换层析和分子筛层析技术,制备得到纯度95%以上的蛋白。通过多次优化结晶条件,获得衍射率约2.1Å的μHD结构域蛋白晶体,为解析其晶体结构奠定了良好的基础。%The human FCHo1 protein (Fer/Cip4 homology domain only proteins 1) is nucleator of clathrin-mediated endocytosis and its C terminalμHD domain ( AP2-μhomology domain) is critical to the correct positioning of FCHo1 in the formation of clathrin-coated vesicles of clathrin-mediated endocytosis.To determine the crystal structure and understand the structural mechanism of human FCHo1μHD domain,it is essential to obtain high resolution protein crystal which can be analyzed by X-Ray.The human FCHo1 μHD was cloned into the expression vector pET15b and verified by bacteria liquid PCR and gene sequencing.Proteins were overexpressed in E. coli BL21 (DE3) which induced by 0.2 mmol/L IPTG at 16℃overnight.The recombined proteins were purified by Ni-affinity chro-matography ( Ni-NTA) , ion exchange chromatography ( IEC) and size exclusion chromatography ( SEC) , respectively.Proteins were detected by SDS-PAGE and the purity was over 95%.Crystal screen kits from Hampton Research were applied to the preliminary screening ofμHD protein by setting-drop method.The human FCHo1μHD protein crystal at about 2.1 Åresolution was acquired after optimizing the condition of crystallization by hanging-drop vapor-diffusion method and data were

  15. Laboratory information management system for membrane protein structure initiative--from gene to crystal.

    Science.gov (United States)

    Troshin, Petr V; Morris, Chris; Prince, Stephen M; Papiz, Miroslav Z

    2008-12-01

    Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.

  16. Gene cloning, expression, and purification of S-layer protein from Bacillus sphaericus%球形芽胞杆菌S层蛋白基因的克隆及原核表达

    Institute of Scientific and Technical Information of China (English)

    杨李; 周鹰; 王运刚; 马桂芳; 崔玉宝

    2012-01-01

    目的 克隆球形芽胞杆菌S层蛋白的编码基因,实现其原核表达并纯化表达产物. 方法 提取球形芽胞杆菌菌体基因组DNA,根据GenBank公布的核酸序列设计并合成引物,扩增S层蛋白编码基因sllB,与pMD19-T载体连接;测序验证后,构建原核表达质粒pET28 a (+)-sllB并转化BL21( DE3)中,经IPTG诱导表达,SDS-PAG和Western blot鉴定表达产物,亲和柱层析纯化重组蛋白. 结果 扩增获得S层蛋白编码基因sllB全长(3 306 bp),与参考序列同源性为97.5%.构建的原核表达质粒pET28 a (+)-sllB转化BL21( DE3)后,SDS-PAGE显示表达蛋白的分子质量为116 ku,与理论值相符.Western blot显示其全蛋白、可溶性蛋白和不溶性蛋白中均被鼠抗Penta-His抗体识别.结论 获得了球形芽胞杆菌S层蛋白编码基因sllB,并实现了其原核表达.%Objective To clone, express and purify surface layer protein from Bacillus sphaericus. Methods Ge-nomic DNA was extracted from B. sphaericus with primers designed in accordance with the nucleotide sequence registered in GenBank. The target gene was ligated to a pMD19-T plasmid. The fragment was then sequenced, 3ub-cloned into the plasmid pET28a ( + ), expressed in E. coli BL21, and purified by nickel-affinity chromatography. Results Full-length sllB cDNA coding for the surface layer protein of Bacillus sphaericus was amplified (3,306 bp) and had 97. 5% homology with the reference sequence (GenBank Accession No. AJ849550). SDS-PAGE and Western blotting showed a specific band of about 116 ku in the whole cell lysate, soluble protein fraction, and insoluble protein fraction. As estimated by chromatography, about 1. 8 mg of the recombinant product was obtained. Conclusion The gene sllB coding for surface layer protein was amplified from Bacillus sphaericus and successfully expressed in E. coli BL21

  17. Expression, crystallization and preliminary X-ray diffraction studies of recombinant Clostridium perfringens β2-toxin

    Energy Technology Data Exchange (ETDEWEB)

    Gurjar, Abhijit A. [Department of Veterinary and Biomedical Science, The Pennsylvania State University (United States); Yennawar, Neela H.; Yennawar, Hemant P. [Macromolecular X-ray Crystallography Facility, The Pennsylvania State University (United States); Rajashankar, Kanagalaghatta R. [Argonne National Laboratory (United States); Hegde, Narasimha V.; Jayarao, Bhushan M., E-mail: bmj3@psu.edu [Department of Veterinary and Biomedical Science, The Pennsylvania State University (United States)

    2007-06-01

    The cloning, expression, purification and crystallization of recombinant Clostridium perfringens β2-toxin is described. The crystals diffracted to 2.9 Å resolution. Clostridium perfringens is a Gram-positive sporulating anaerobic bacterium that is responsible for a wide spectrum of diseases in animals, birds and humans. The virulence of C. perfringens is associated with the production of several enterotoxins and exotoxins. β2-toxin is a 28 kDa exotoxin produced by C. perfringens. It is implicated in necrotic enteritis in animals and humans, a disease characterized by a sudden acute onset with lethal hemorrhagic mucosal ulceration. The recombinant expression, purification and crystallization of β2-toxin using the batch-under-oil technique are reported here. Native X-ray diffraction data were obtained to 2.9 Å resolution on a synchrotron beamline at the F2 station at Cornell High Energy Synchrotron Source (CHESS) using an ADSC Quantum-210 CCD detector. The crystals belong to space group R3, with a dimer in the asymmetric unit; the unit-cell parameters are a = b = 103.71, c = 193.48 Å, α = β = 90, γ = 120° using the hexagonal axis setting. A self-rotation function shows that the two molecules are related by a noncrystallographic twofold axis with polar angles ω = 90.0, ϕ = 210.3°.

  18. Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

    Science.gov (United States)

    Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

    2015-01-01

    Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries.

  19. 岩栖蝮蛇类凝血酶纯化、cDNA克隆和序列分析%Purification, cDNA cloning and sequence analysis of thrombin-like enzyme from Gloydius saxatilis

    Institute of Scientific and Technical Information of China (English)

    孙德军; 杨春伟; 杨同书; 颜炜群; 王伟

    2003-01-01

    Thrombin-like enzyme has great medical application in treating thrombus. A thrombin-like enzyme from Gloydius saxatilis snake venom was isolated and purified to homogeneity by a rapid and effective method using ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on heparin-sepharose.SDS-polyacrylamide electrophoresis under reducing condition revealed that the purified enzyme had a single protein band and its molecular weight was 32000 dalton.Total RNAs were extracted from the venom gland of the G.saxatilis snake.Using degenerate primers,we amplified the cDNA of the thrombin-like enzyme gene in the venom gland of G.saxatilis using the reverse transcription-polymerase chainreaction (RT-PCR) method.The cDNA fragment was inserted into pGEMT vector,cloned and its nucleotide sequence was determined.Its open reading frame is composed of 774 nucleotides and codes a protein prezymogen of 258 amino acids,including a putative secretory signal peptide of 18 amino acids and a proposed pro-peptide of 6 amino acid residues.It contains 12 cysteine residues.The sequence analysis indicates that the deduced amino acid sequence of the cDNA fragment shares high identity with the thrombin-like enzyme genes of other snakes in the gene bank.The query sequence exhibits strong amino acid sequence homology of 88%,88% and 86% to the serine proteas of T.gramineus,thrombin-like defibrase Ⅰ of D.acutus and serine protease catroxase Ⅱ of C.atrox respectively.Based on the amino acid sequences of other thrombin-like enzymes,the catalytic residues and disulfide bridges of this thrombin-like enzyme are deduced as follows:catalytic residues,His65,Asp110,Ser%204;and six disulfide bridges Cys31-Cys163,Cys50-Cys66,Cys98-Cys256,Cys142-Cys210,Cys174-Cys189 and Cys200-Cys225.According to the possible linked glycosylation sites N-X-T (Asn-X-Thr) or N-X-S (Asn-X-Ser),its possible glycosylation sites are N44-S45-T46 and N251-T252-T253 residues [Acta Zoologica Sinica 49(6):878-882,2003].

  20. 化脓性链球菌脂蛋白FtsB的基因克隆、达、纯化与功能研究%Cloning, expression, purification and functional study of lipoprotein FtsB from Streptococcus pyogenes

    Institute of Scientific and Technical Information of China (English)

    李慧; 吕洁; 王红翠; 王南杰; 李楠; 詹胜; 阳小燕; 孙雪松

    2011-01-01

    体外克隆化脓性链球菌5005的tsb基因,表达并纯化FtsB蛋白,并对其铁色素结合特性进行初步研究.首先通过PCR方法从基因组中扩增ftsb基因,将其连接到pGEX4T-1载体上,转入大肠杆菌DH5a中大量扩增,将测序正确的重组载体转化到表达宿主大肠杆菌BL21进行表达,优化诱导表达条件.利用亲和层析方法纯化表达产物,并对FtsB的铁色素结合特性进行研究,同时通过DEPC封闭组氨酸实验对其结合位点进行初步研究.成功构建原核表达载体pGEX-ftsb,获得分子量约33 kD的野生型FtsB蛋白,产率为30 mg/L.紫外-可见光谱研究表明FtsB和铁色素结合后在450 nm处出现紫外吸收峰,DEPC封闭组氨酸实验证明FtsB中组氨酸不参与铁色素的结合.对FtsB蛋白进行铁色素结合特性和结合位点进行研究,为进一步研究细菌中的铁色素转运机理及开发疫苗候选物和药靶奠定一定的理论基础.%This study aims to clone ftsb gene from Streptococcus pyogenes 5005, to express and purify FtsB, and to characterize its ferrichrome binding properties in vitro. DNA fragments encoding ftsb was obtained by PCR and inserted into plasmid pGEX4T-l, followed by transformation into Escherichia coli DH5a. The constructed recombinant plasmid was transformed into Escherichia coli BL21 to express FtsB. The conditions used to induce expression were optimized. The protein was purified using affinity chromatography. The ferrichrome binding property was initially characterized, and the binding site was investigated by blocking histidine residues, with DEPC. The 33 kD FtsB was successfully expressed and purified, and the yield was 30 mg/L. The UV-visible spectra showed the FtsB had a visible absorption peak at 425 nm with ferrichrome and that histidine didn't participate in ferrichrome binding. This approach reports the ferrichrome binding properties and binding site of FtsB, providing theoretical foundations for the further study of

  1. 华支睾吸虫mGST新基因的克隆、表达与纯化%CLONING, EXPRESSION AND PURIFICATION OF A NOVEL GENE OF mGST FROM CLONORCHIS SINENSIS

    Institute of Scientific and Technical Information of China (English)

    伍忠銮; 吴德; 吴忠道; 徐劲; 余新炳

    2005-01-01

    目的扩增华支睾吸虫一个微粒体谷胱甘肽转移酶(mGST)新基因,明确是否存在内含子,并进行原核表达与纯化. 方法用PCR方法分别从华支睾吸虫成虫cDNA(质粒)文库和基因组DNA中扩增mGST基因并测序,将编码基因定向克隆到原核表达载体pET-30a(+),在大肠埃希菌BL21/DE3中表达,按照Ni-NTA agarose说明书纯化重组蛋白,用SDS-PAGE和TLC分析. 结果 mGST基因全长486 bp,没有内含子,构建的原核表达质粒pET-30a(+)-mGST在大肠埃希菌中得到了有效表达,融合蛋白的分子质量单位约为24 ku.重组蛋白达细菌总蛋白质的11%,纯化后的重组蛋白纯度为83%. 结论 mGST基因没有内含子,在大肠埃希菌中能有效表达,得到了纯化的重组蛋白,为进一步研究其功能奠定了基础.%Objective To amplify a new encoding gene of microsomal glutathione transferase (mGST) from adult Clonorchis sinensis, determine whether it had intron or not, express it in Escherichia coli and purify the recombinant protein. Methods The gene encoding mGST was obtained from the cDNA (plasmid) library and genomic DNA of adult C. sinensis by PCR and sequenced, respectively. The coding gene was cloned into the prokaryotic expression vector pET-30a(+) and expressed in E. coli BL21/DE3. The recombinant protein was purified according to the protocol of Ni-NTA agarose (QIAGEN, Germany). It was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thin-layer chromatography (TLC). Results The full length of the gene encoding mGST was 486 bp, it had no intron. The recombinant plasmid pET-30a(+)-mGST was efficiently expressed in E. coli, its molecular weight was about 24 ku. The recombinant protein reached up to 11% of total bacterial protein. The purity of the purified recombinant protein was 83%. Conclusion The mGST gene of C. sinensis has no intron. Its efficient expression has been achieved in E. coli. The recombinant protein was purified

  2. 假坚强芽孢杆菌丙氨酸脱氢酶的原核表达纯化与结晶%Prokaryotic Expression,Purification and Crystallization of Alanine Dehydrogenase in Bacillus pseudofirmus

    Institute of Scientific and Technical Information of China (English)

    唐昭娜; 张翠英; 胡平雄; 易秋分; 杨江丽; 董辉

    2015-01-01

    丙氨酸脱氢酶(alanine dehydrogenase,Ald)是一种NAD+依赖性的氨基酸脱氢酶,能可逆地催化丙氨酸氧化脱氨生成丙酮酸和氨.实验以假坚强芽孢杆菌的丙氨酸脱氢酶为研究对象,将目的基因克隆到 pET-22,b(+)原核表达载体上,并在大肠杆菌中完成蛋白的高效表达.通过镍离子亲和层析、离子交换层析和凝胶过滤层析等纯化方法,得到了高纯度的目的蛋白.利用气相扩散法对目的蛋白进行结晶,最终得到分辨率为0.31,nm的蛋白晶体.%Alanine dehydrogenase(Ald)is a NAD+-dependent amino acid dehydrogenase. It catalyzes the reversible oxida-tive deamination of L-alanine to pyruvate and ammonia. An Ald gene from Bacillus pseudofirmus was cloned into the pro-karyotic expression vector pET-22,b(+)and the Ald protein was overexpressed inEscherichia coli. The Ald protein was purified by Ni2+-chelating affinity chromatography,anion-exchange chromatography and gel filtration chromatography. Crystals were grown with the vapour-diffusion method and diffracted to 0.31,nm resolution.

  3. 蓝氏贾第鞭毛虫沉默信息调节因子2(Sir2)基因的克隆、表达与纯化%Cloning, Expression and Purification of Silent Information Regulator 2 from Giardia lamblia

    Institute of Scientific and Technical Information of China (English)

    张霞; 巨红妹; 王云华; 李雅杰

    2011-01-01

    Objective To clone and express silent information regulator 2 (Sir2) gene from Giardia lamblia Methods The GZSir2 gene was amplified by PCR from genomic DNA of Giardia lamblia (Chinese strain C2 clone). PCR product was cloned into pMD-19T vector and transformed into K coli JM109. The recombinant plasmid was sequenced and then cloned into the pET28b vector. The pET28b-G/lSir2 recombinant plasmid was transformed into E. Coli BL21(DE3), followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE. Inclusion bodies were dissolved with 8 mol/L urea, and the supernatant was collected and applied to Ni2* affinity chromatography. The purified recombinant protein was renatured by dialysis and verified by Western blotting using anti-His tag antibody. Results G£>ir2 gene sequence was cloned. The GZSir2 open reading frame (1680bp) encoded a 559-amino acid protein with M, 62 800. The recombinant plasmid pET28b-G/Sir2 expressed an inclusion body protein of G/Sir2 after being induced with IPTG. The protein purity reached above 80% after purification. The purified protein was renatured by dialysis. The recombinant G/Sir2 was recognized by anti-His tag antibody. Conclusion The coding sequence of GZSir2 gene was cloned and expressed in vitro. The recombinant protein was identified by anti-His tag antibody.%目的 获取蓝氏贾第鞭毛虫(Giardia lamblia,Gl)沉默信息调节因子2(Sir2)基因序列及其重组蛋白.方法 以蓝氏贾第鞭毛虫中国C2克隆株基因组DNA为模板,PCR扩增GlSir2基因编码序列,将所得片段连接至pMD-19T质粒.转化大肠埃希菌(E.coli)JM109,挑取阳性克隆进行测序.将GlSir2基因插入原核表达载体pET28b,构建表达载体pET28b-GlSir2,转化E.coli BL21(DE3),用异丙基硫代半乳糖苷(IPTG)诱导表达.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白的表达情况.收集重组表达产物,用8 mol/L尿素溶解包涵体并收集上清,镍

  4. Crystallization and preliminary X-ray crystallographic analysis of zebrafish prototype galectin Drgal1-L2.

    Science.gov (United States)

    Scott, Stacy A; Cozier, Matthew O; Dubar, Pauline D I; Ramakrishna, Manasa; Scott, Ken; Blanchard, Helen

    2010-12-01

    Zebrafish (Danio rerio) are an important developmental and embryological model given the optical clarity of the embryos and larvae, which permits real-time viewing of developing pathologies. More recently, a broader scope for these vertebrates to model a range of human diseases, including some cancers, has been indicated. Zebrafish Drgal1-L2 has been identified as an orthologue of mammalian galectin-1, which is is a carbohydrate-binding protein that exhibits β-galactoside-binding specificity and which is overexpressed by many aggressive human cancers. This study describes the cloning, expression in Escherichia coli, purification and crystallization of recombinant Drgal1-L2 protein in the presence of lactose (ligand). X-ray diffraction data from these novel crystals of zebrafish Drgal1-L2 were collected to a resolution of 1.5 Å using a synchrotron-radiation source, enabling their characterization.

  5. The Clone Factory

    Science.gov (United States)

    Stoddard, Beryl

    2005-01-01

    Have humans been cloned? Is it possible? Immediate interest is sparked when students are asked these questions. In response to their curiosity, the clone factory activity was developed to help them understand the process of cloning. In this activity, students reenact the cloning process, in a very simplified simulation. After completing the…

  6. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.

    Science.gov (United States)

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective.

  7. Cloning, Expression and Purification of Ultraspiracle Protein Gene from Apolygus lucorum%绿盲蝽超气门蛋白ALUSP的克隆、原核表达及纯化

    Institute of Scientific and Technical Information of China (English)

    谭永安; 肖留斌; 孙洋; 赵静; 柏立新

    2014-01-01

    约65 kD的蛋白,并且该重组ALUSP蛋白主要以包涵体形式存在;收集含有目的基因的菌株进行GST琼脂糖亲和层析和分子筛层析,最后进行复性和纯化后在65 kD附近仅出现1条清晰的特异性条带,表明已得到纯化的靶蛋白。【结论】克隆了ALUSP全长,其具有昆虫超气门蛋白的典型特征,并获得了原核表达的重组蛋白。%[Objective] The objectives of this study are to clone the ultraspiracle protein gene of Apolygus lucorum (ALUSP) and obtain the recombinant protein of prokaryotic expression, and to reveal the function of ALUSP and offer a basis for research of the ecdysis and metamorphosis of A. lucorum. [Method] The conserved sequence of A. lucorum was obtained by using the degenerate primers from the known USP genes of the other insects. A new set of gene-specific primers matching the primers in the 3′-and 5′-full RACE kit were designed and the original sequence files were assembled to obtain the complete cDNA sequence of the ALUSP. The characteristics of ALUSP gene sequences and which coding proteins were analyzed by using the bioinformatics methods, and the phylogenetic tree was also constructed. To construct its prokaryotic expression vector, T-vector containing ALUSP was dual-enzyme digested by EcoR I and Xho I. The expression vector was induced to express at 15, 25, 30 and 37℃. ALUSP purified protein gene in functional areas was gotten by the GST agarose affinity chromatography and molecular sieve chromatography.[Result]The open reading frame of ALUSP was 1 005 bp in length, encoding a 334 amino acid polypeptide with a predicted molecular weight of 56.63 kD and the theoretical isoelectric point of 8.42. Protein signature analysis revealed that the protein encoded by ALUSP shared typical structural features of ultraspiracle proteins with other insects, including A/B domain (249 bp), C domain (198 bp), D domain (69 bp) and E domain (489 bp), respectively. C domain was

  8. Gas purification process

    Energy Technology Data Exchange (ETDEWEB)

    Hoelter, H.; Gresch, H.; Igelbuescher, H.; Dewert, H.

    1987-08-13

    To avoid the problems of reheating in a wet process as well as the problems of higher gas supply in a dry process, the invention proposes to separate the raw gas in two component currents, one of which undergoes wet purification while the other is led through a dry purification process. The two component currents are mixed before entering the stack. The dry chemisorption masses added in substoichiometric doses are treated in a milk-of-lime processing stage, after which the reacted and non-reacted chemisorption masses are treated by wet purification and then by oxidation.

  9. The Borexino purification system

    Science.gov (United States)

    Benziger, Jay

    2014-05-01

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

  10. Cloning of observables

    OpenAIRE

    Ferraro, Alessandro; Galbiati, Matteo; Paris, Matteo G. A.

    2005-01-01

    We introduce the concept of cloning for classes of observables and classify cloning machines for qubit systems according to the number of parameters needed to describe the class under investigation. A no-cloning theorem for observables is derived and the connections between cloning of observables and joint measurements of noncommuting observables are elucidated. Relationships with cloning of states and non-demolition measurements are also analyzed.

  11. OPTIMASI KONDISI PEMURNIAN ASAM LEMAK OMEGA-3 DARI MINYAK HASIL SAMPING PENEPUNGAN TUNA (Thunnus sp DENGAN KRISTALISASI UREA [Optimizing Conditions for the Purification of Omega-3 Fatty Acids from By-product of Tuna (Thunnus sp Meal Processing by Urea Crystallization

    Directory of Open Access Journals (Sweden)

    Teti Estiasih1*

    2009-12-01

    Full Text Available Omega-3 fatty acids (-3 are proven to have health beneficial effects. Some effort had been done to obtained oil high in -3 fatty acids. Among the methods developed, urea crystallization was preferred because it is simple, economic, and result in high purity of fatty acids. A source that had not been widely explored for -3 fatty acids production is the by-product of tuna meal processing. This research studied the optimization condition for separation and purification of -3 fatty acids from the by-product of tuna meal processing by urea crystallization. Crystallization reaction conditions of urea inclusion were optimized using the response surface methodology, and the model was developed.Optimization result showed a quadratic polynomial regression equation of Y= 140,52677X1 + 8,38203X2 – 19,85850X12 – 0,12173X22 – 0,74000X1X2 – 240,33546 with X1=urea to fatty acid ratio and X2=crystallization time. Maximum response was obtained at urea to fatty acid ratio of 3,07:1,crystallization time of 25,10 hours, and predicted response was 80,60%. Analysis of variance showed that urea to fatty acid ratio and crystallization time affected response. Under optimal conditions, the product was 3.89 times concentrated and the purity of -3 fatty acids was 81,98%. Verification result revealed that the predicted value from this model was reasonably close to the experimentally observed value. Urea crystallization process changed quality parameters that were oxidation level (peroxide value, anisidin value, and totox value, Fe and Cu content, P content, and water content. The changes were caused by adsorption of primary and secondary oxidation products, part of metals, and phosphoric compound onto unsaponifiable matters or soluble into water.

  12. Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W., E-mail: n.isaacs@chem.gla.ac.uk [Department of Chemistry and WestChem, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

    2007-07-01

    P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6{sub 1}22, with unit-cell parameters a = b = 123.27, c = 134.43 Å.

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

    Science.gov (United States)

    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.

  14. 苏云金芽胞杆菌WB5中营养期杀虫蛋白3Aa基因(vip3Aa)的克隆、表达与纯化%Cloning, Expression and Purification of Vegetative Insecticidal Protein 3Aa Gene(vip3Aa) from Bacillus thuringiensis WB5

    Institute of Scientific and Technical Information of China (English)

    陈凡冰; 宋飞飞; 石鹏; 黄志鹏; 关雄

    2014-01-01

    sequencing and NdeⅠ/XbaⅠdigested analysis. Using Universal DNA Purification Kit, an approximate 2.4 kb DNA product was purified from NdeⅠ/XbaⅠdigested pMD18-T-vip3Aa, and then inserted into multiple cloning sites of the expression vector pCzn1 to generate the recombinant expression vector pCzn1-vip3Aa. The inserted fragment and its reading frame were confirmed by NdeⅠ/XbaⅠdigestion and nucleotide sequence analysis. pCzn1-vip3Aa was transformed into E. coli ArcticExpressTM (DE3) and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). SDS-PAGE showed that the relative molecular weight of the expressed protein was about 88 kD in both supernatant and precipitated. In addition, the expressed protein was purified by Ni-NTA affinity chromatography. The success of the subcloned vip3Aa gene and the expression and purification of Vip3Aa provide basic data for furthur research on its action receptor in insect mid gut.

  15. Molecular cloning and in silico studies of physiologically significant trehalase from Drosophila melanogaster.

    Science.gov (United States)

    Shukla, Ekta; Thorat, Leena; Bhavnani, Varsha; Bendre, Ameya D; Pal, J K; Nath, B B; Gaikwad, S M

    2016-11-01

    Trehalase, a physiologically important glycosidase is known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a gene fragment, encoding the catalytically active trehalase from Drosophila melanogaster (DmTre) and its heterologous expression in Escherichia coli. The 1275bp gene was overexpressed in two different vectors viz., pET28a and pCOLD TF and investigated for variable soluble expression, purification and activity of the recombinant enzyme with optimum pH and temperature of enzyme as 6 and 55°C, respectively. The sequence was characterized in silico by subjecting it to homology search, multiple sequence alignment and phylogenetic tree construction revealing its identity to other trehalases which belong to glycoside hydrolase family 37. The deduced amino acid sequence and modeled 3D structure of DmTre possessed all features of trehalase superfamily, including signature motifs and catalytic domain. The active site pocket of recombinant DmTre was compared with the crystal structure of E. coli trehalase identifying Glu424 and Asp226 as the putative catalytic residues. Additionally, enzyme-substrate docking suggests possible involvement of other residues in the catalysis along with Asp226. The present study holds significance in understanding the structural aspects of Drosophila trehalase in spite of unavailabilty of eukaryotic trehalase crystal structure.

  16. Statement on Human Cloning

    Science.gov (United States)

    ... ban on efforts to implant a human cloned embryo for the purpose of reproduction. The scientific evidence ... stem cell research, including the use of nuclear transplantation techniques (also known as research or therapeutic cloning), ...

  17. Ethical issues in cloning.

    Science.gov (United States)

    Satris, S

    2000-01-01

    There is great public concern with the ethics of human cloning. This paper briefly examines some of what I identify as pseudo-problems or myths associated with cloning, and some of the more substantial ethical concerns.

  18. Cloning, purification and characterisation of Staphylococcus warneri lipase 2

    NARCIS (Netherlands)

    van Kampen, M.D.; Rosenstein, R.; Götz, F.; Egmond, M.R.

    2010-01-01

    A gene encoding an extracellular lipase was identified in Staphylococcus warneri 863. The deduced lipase is organised as a prepro-protein and has significant similarity to other staphylococcal lipases. The mature part of the lipase was expressed with an N-terminal histidine tag in Escherichia coli,

  19. Cloning of nis gene and Nisin purification from Lactococcus lactis ...

    African Journals Online (AJOL)

    user

    2014-12-31

    Dec 31, 2014 ... Author(s) agree that this article remains permanently open access under the terms of the ... 2005). Cultivation of pathogenic strains. Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, .... onto the top of separating gel where the comb was inserted. The ..... Wolf CE, Gibbons WR (1995).

  20. Cloning, expression and purification of cold adapted acetate kinase ...

    African Journals Online (AJOL)

    Yomi

    2012-04-10

    Apr 10, 2012 ... The molecular mass of the enzyme is about 86 kDa, which is .... 540 nm using a micro plate reader (Bio-Rad, model 680XR). A ... The maximum amount of soluble acetate kinase was ..... A consensus view of protein dynamics.

  1. Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins

    Science.gov (United States)

    2005-01-01

    acquired after brucellosis is characterized by fever, chills, malaise, ingestion of foodstuffs, especially unpasteurized dairy and a chronic course...synthesis of OMP genes of B. suis. Seven OMP genes of the samples were incubated with 1:2000 secondary goat B, suis were PCR synthesized using...antibody and protects rhesus macaques from infectious brucellosis induced by subsequent aerosolFig. 3. PCR detection of pET-DEST42-B, suis OMP genes

  2. Quick and clean cloning.

    Science.gov (United States)

    Thieme, Frank; Marillonnet, Sylvestre

    2014-01-01

    Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

  3. Biotechnology Protein Expression and Purification Facility

    Science.gov (United States)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  4. Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    DEFF Research Database (Denmark)

    Lundqvist, Magnus; Edfors, Fredrik; Sivertsson, Åsa

    2015-01-01

    We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We...... at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations....

  5. Statement on Human Cloning

    Science.gov (United States)

    ... as our understanding of this technology advances. Support Stem Cell Research (including Research Cloning) AAAS supports stem cell research, including the use of nuclear transplantation techniques (also ...

  6. Cloning and expression of aequorin photoprotein using intein tag

    Directory of Open Access Journals (Sweden)

    Elah sadat Seyed Hosseini

    2015-01-01

    Full Text Available Background: Intein (INT, is the internal parts of the protein which can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. This protein sequence and their characteristic of self-cleavage by thiol induction, temperature and pH changes is used for protein purification. The advantage of this method compared to the other protein purification methods is that it doesn’t require any protease enzyme and protease removal steps that make this method important economically. In this study, aequorin photoprotein was hybridized with INT in molecular form and its expression was evaluated. Materials and Methods: In this study, aequorin coding gene that was cloned in pET21-a in the previous studies, was cloned in pTYB21 vector containing INT tag by specific primers and restriction enzymes. Then the resulting pTY-aequarin was transformed to the ER2566 expression strain and cloning accuracy was confirmed by electrophoresis, western blotting and sequencing. Results: The photoprotein aequorin was cloned into SapI/PstI restriction site of pTYB21 plasmid accurately and successfully. Aequorin- INT hybrid protein expression confirmed using traditional methods. Conclusion: The photoprotein aequorin constract in fused with INT confirmed by molecular methods. Also rate of Aequorin- INT expression determined about %25 of cell total protein.

  7. Purification, crystallization and preliminary X-ray structural studies of a 7.2 kDa cytotoxin isolated from the venom of Daboia russelli russelli of the Viperidae family

    Energy Technology Data Exchange (ETDEWEB)

    Roy Choudhury, Subhasree [Department of Physiology, University of Calcutta, 92 Acharya Prafulla Chandra Road, Kolkata 700 009 (India); Gomes, Aparna [New Drug Development Division, Indian Institute Of Chemical Biology, 4 Raja S. C. Mallick Road, Kolkata 700 032 (India); Gomes, Antony [Department of Physiology, University of Calcutta, 92 Acharya Prafulla Chandra Road, Kolkata 700 009 (India); Dattagupta, Jiban K.; Sen, Udayaditya, E-mail: udayaditya.sen@saha.ac.in [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064 (India); Department of Physiology, University of Calcutta, 92 Acharya Prafulla Chandra Road, Kolkata 700 009 (India)

    2006-03-01

    A cytotoxin from Indian Russell’s viper (D. russelli russelli) venom having multifunctional activity has been crystallized in space group P4{sub 1}. Larger crystals diffracted to 1.5 Å but were found to be twinned; preliminary data were therefore collected (2.93 Å) from a smaller crystal. A cytotoxin (MW 7.2 kDa) from Indian Russell’s viper (Daboia russelli russelli) venom possessing antiproliferative activity, cardiotoxicity, neurotoxicity and myotoxicity has been purified, characterized and crystallized. The crystals belong to the tetragonal space group P4{sub 1}, with unit-cell parameters a = b = 47.94, c = 50.2 Å. Larger crystals, which diffracted to 1.5 Å, were found to be twinned; diffraction data were therefore collected to 2.93 Å resolution using a smaller crystal. Molecular-replacement calculations identified two molecules of the protein in the asymmetric unit, which is in accordance with the calculated V{sub M} value.

  8. Electron beam silicon purification

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-11-15

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

  9. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    Science.gov (United States)

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  10. Cloning-free CRISPR

    NARCIS (Netherlands)

    Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I

    2015-01-01

    We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each targ

  11. Cloning-free CRISPR

    NARCIS (Netherlands)

    Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I

    2015-01-01

    We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each

  12. Cloning-free CRISPR

    NARCIS (Netherlands)

    Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels|info:eu-repo/dai/nl/194303403; Sherwood, Richard I

    2015-01-01

    We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each targ

  13. Practice and design of the self-purification system for heavy metals-bearing contaminants

    Institute of Scientific and Technical Information of China (English)

    Qian Guangren

    2008-01-01

    Many minerals in nature have self-purification capacity to hold and stabilize deleterious contaminants into their lattice structures,which can be used for treatment of heavy metals-bearing contaminants.Hydrotalcite Layer Double Hy-droxide(LDH),tobermorite Calcium Silicate Hydrate(CSH)and apatite are ubiquitous minerals in nature,having higher geochemical stability and potential for binding and stabilizing heavy metals.Based on the elucidation of crystal structure property and self-purification principles of the three minerals above,this article discussed how to design the self-purification system of heavy metal-bearing contaminants.

  14. Crystal growth of pure and impurity-doped lead chloride single crystals from the melt

    NARCIS (Netherlands)

    Willemsen, B.

    An account is given of the purification and the crystal growing of lead chloride. Difficulties observed are blackening of the material and the formation of oxides. It is evident that oxygen should be avoided. The influence of water vapour is studied with mass spectroscopy, crystal microbalance

  15. Crystal growth of pure and impurity-doped lead chloride single crystals from the melt

    NARCIS (Netherlands)

    Willemsen, B.

    1971-01-01

    An account is given of the purification and the crystal growing of lead chloride. Difficulties observed are blackening of the material and the formation of oxides. It is evident that oxygen should be avoided. The influence of water vapour is studied with mass spectroscopy, crystal microbalance measu

  16. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  17. Expression, purification, crystallization, data collection and preliminary biochemical characterization of methicillin-resistant Staphylococcus aureus Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase

    Energy Technology Data Exchange (ETDEWEB)

    Seetharamappa, Jaldappagari [Scottish Structural Facility and Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST,Scotland (United Kingdom); Department of Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, Karnataka State (India); Oke, Muse; Liu, Huanting; McMahon, Stephen A.; Johnson, Kenneth A.; Carter, Lester; Dorward, Mark; Zawadzki, Michal [Scottish Structural Facility and Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST,Scotland (United Kingdom); Overton, Ian M.; Niekirk, C. A. Johannes van [Scottish Structural Facility and School of Life Sciences Research, University of Dundee, Dow Street, Dundee DD1 5EH,Scotland (United Kingdom); Graham, Shirley; Botting, Catherine H.; Taylor, Garry L.; White, Malcolm F. [Scottish Structural Facility and Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST,Scotland (United Kingdom); Barton, Geoffrey J. [Scottish Structural Facility and School of Life Sciences Research, University of Dundee, Dow Street, Dundee DD1 5EH,Scotland (United Kingdom); Coote, Peter J.; Naismith, James H., E-mail: naismith@st-andrews.ac.uk [Scottish Structural Facility and Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST,Scotland (United Kingdom)

    2007-05-01

    As part of work on S. aureus, the crystallization of Sar2028, a protein that is upregulated in MRSA, is reported. Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase with a molecular weight of 48 168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 Å, α = β = γ = 90°. Analysis of the systematic absences along the three principal axes indicated the space group to be P2{sub 1}2{sub 1}2{sub 1}. A complete data set was collected to 2.5 Å resolution.

  18. Air/Water Purification

    Science.gov (United States)

    1992-01-01

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

  19. Water Purification Product

    Science.gov (United States)

    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.

  20. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  1. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

  2. Unified Approach to Universal Cloning and Phase-Covariant Cloning

    OpenAIRE

    Hu, Jia-Zhong; Yu, Zong-Wen; Wang, Xiang-Bin

    2008-01-01

    We analyze the problem of approximate quantum cloning when the quantum state is between two latitudes on the Bloch's sphere. We present an analytical formula for the optimized 1-to-2 cloning. The formula unifies the universal quantum cloning (UQCM) and the phase covariant quantum cloning.

  3. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  4. Eukaryotic expression, purification, identification, and tissuedistribution of porcine PID1

    OpenAIRE

    Chen, Xiaoling; Wang, Huan; ZHOU, BO; Huang, Zhiqing; Jia, Gang; Liu, Guangmang; Zhao, Hua

    2015-01-01

    Abstract: Phosphotyrosine interaction domain containing 1 (PID1) is a recently discovered gene related to lipid metabolism and may play an important role in fat deposition. In this study, in order to scale up the production of the active recombinant porcine PID1 (pPID1) protein, we reported the expression and purification of a His-tagged version of pPID1 in the yeast Pichia pastoris. The pPID1 cDNA was cloned into the pPICZαA vector and was expressed in methylotrophic yeast (P. ...

  5. Main: Clone Detail [KOME

    Lifescience Database Archive (English)

    Full Text Available Clone Detail Mapping Pseudomolecule data detail Detail information Mapping to the TIGR japonica Pseudomolecu...les kome_mapping_pseudomolecule_data_detail.zip kome_mapping_pseudomolecule_data_detail ...

  6. BIOETHICS AND HUMAN CLONING

    Directory of Open Access Journals (Sweden)

    Željko Kaluđerović

    2011-12-01

    Full Text Available In this paper the authors analyze the process of negotiating and beginning of the United Nations Declaration on Human Cloning as well as the paragraphs of the very Declaration. The negotiation was originally conceived as a clear bioethical debate that should have led to a general agreement to ban human cloning. However, more often it had been discussed about human rights, cultural, civil and religious differences between people and about priorities in case of eventual conflicts between different value systems. In the end, a non-binding Declaration on Human Cloning had been adopted, full of numerous compromises and ambiguous formulations, that relativized the original intention of proposer states. According to authors, it would have been better if bioethical discussion and eventual regulations on cloning mentioned in the following text had been left over to certain professional bodies, and only after the public had been fully informed about it should relevant supranational organizations have taken that into consideration.

  7. Do Managers Clone Themselves?

    Science.gov (United States)

    Baron, Alma S.

    1981-01-01

    A recent questionnaire survey provides statistics on male managers' views of female managers. The author recommends that male managers break out of their cloning behavior and that the goal ought to be a plurality in management. (Author/WD)

  8. Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification.

    Science.gov (United States)

    Fujii, Yuki; Kaneko, Mika K; Ogasawara, Satoshi; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Honma, Ryusuke; Kato, Yukinari

    2017-04-01

    Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the "RAP tag" system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification.

  9. Effect of charcoal on water purification

    OpenAIRE

    Suzuki, Hirotaka; Kawahigashi, Tatsuo

    2014-01-01

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

  10. Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490(963-1138) domain of TamB from Escherichia coli.

    Science.gov (United States)

    Josts, Inokentijs; Grinter, Rhys; Kelly, Sharon M; Mosbahi, Khedidja; Roszak, Aleksander; Cogdell, Richard; Smith, Brian O; Byron, Olwyn; Walker, Daniel

    2014-09-01

    TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963-1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Å resolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future.

  11. Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated from Sulfolobus tokodaii strain 7

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, Noboru; Kumarevel, Thirumananseri, E-mail: tskvel@spring8.or.jp; Matsunaga, Emiko; Shinkai, Akeo [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Kuramitsu, Seiki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Department of Biological Sciences, Graduate School of Science, Osaka University, Tayonaka, Osaka 560-0043 (Japan); Yokoyama, Shigeyuki, E-mail: tskvel@spring8.or.jp [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2007-11-01

    A putative member of the Lrp/AsnC family of transcriptional regulators, ST1022 from S. tokodaii strain 7, has been purified and crystallized in the absence and presence of the effector l-glutamine. A molecular-replacement solution was found using the FL11 transcriptional regulator from Pyrococcus sp. OT3 as a model and structural refinement is under way. The Lrp/AsnC family of transcriptional regulators, also known as feast/famine transcriptional regulators, are widely distributed among bacteria and archaea. This family of proteins are likely to be involved in cellular metabolism, with exogenous amino acids functioning as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of ST1022, a member of the Lrp/AsnC family of proteins, is reported with and without exogenous glutamine as the effector molecule. The crystals of native ST1022 and of the putative complex belong to the tetragonal space group I422, with unit-cell parameters a = b = 103.771, c = 73.297 Å and a = b = 103.846, c = 73.992 Å, respectively. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of one monomer per asymmetric unit.

  12. Purification, crystallization and preliminary X-ray analysis of the galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) of the ABC transporter from Bifidobacterium longum JCM1217

    Energy Technology Data Exchange (ETDEWEB)

    Wada, Jun [Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Suzuki, Ryuichiro; Fushinobu, Shinya [Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kitaoka, Motomitsu [National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642 (Japan); Wakagi, Takayoshi; Shoun, Hirofumi [Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Ashida, Hisashi [Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Kumagai, Hidehiko; Katayama, Takane, E-mail: takane@ishikawa-pu.ac.jp [Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836 (Japan); Yamamoto, Kenji [Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan)

    2007-09-01

    Preliminary X-ray analysis of the galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) of the ABC transporter from B. longum is described. A recombinant galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) from Bifidobacterium longum JCM1217 has been prepared and crystallized by the hanging-drop vapour-diffusion method using 10 mg ml{sup −1} purified enzyme, 0.01 M zinc sulfate, 0.1 M MES buffer pH 5.9–6.4 and 20–22%(v/v) PEG MME 550 in the presence of 5 mM disaccharide ligands. Suitable crystals grew after 10 d incubation at 293 K. The crystals belong to space group C222{sub 1}, with unit-cell parameters a = 106.3, b = 143.6, c = 114.6 Å for the lacto-N-biose I complex and a = 106.4, b = 143.4, c = 115.5 Å for the galacto-N-biose complex, and diffracted to 1.85 and 1.99 Å resolution, respectively.

  13. Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Chaurasia, Priyanka; von Ossowski, Ingemar; Palva, Airi; Krishnan, Vengadesan

    2015-01-01

    SpaD is the predicted backbone-pilin subunit of the SpaFED pilus, whose loci are encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG, a Gram-positive gut-adapted commensal strain with perceived probiotic benefits. In this study, soluble recombinant SpaD protein was overproduced in Escherichia coli and then purified by Ni2+-chelating affinity and gel-filtration chromatography. After limited proteolysis with α-chymotrypsin, good-quality crystals of SpaD were obtained which diffracted beyond 2.0 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=50.11, b=83.27, c=149.65 Å. For phasing, sodium iodide-derivatized crystals were prepared using the halide quick-soaking method and diffraction data were collected in-house to a resolution of 2.2 Å. An interpretable electron-density map was successfully obtained using single-wavelength anomalous diffraction (SAD).

  14. Crystallization and X-ray diffraction analysis of the CH domain of the cotton kinesin GhKCH2

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Xinghua [China Agricultural University, No. 2 Yuanmingyuanxilu, Haidian District, Beijing 100094, People’s Republic of (China); The Fourth Military Medical University, No. 169 Changlexi Road, Xincheng District, Xi’an 710032, People’s Republic of (China); Chen, Ziwei; Li, Ping; Liu, Guoqin, E-mail: liu@cau.edu.cn [China Agricultural University, No. 2 Yuanmingyuanxilu, Haidian District, Beijing 100094, People’s Republic of (China)

    2016-02-19

    The cloning, expression, purification and crystallization of the CH domain of the plant-specific kinesin GhKCH2 is reported. GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (K{sub d} = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30–166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystals diffracted to a resolution of 2.5 Å and belonged to space group P2{sub 1}, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å{sup 3} Da{sup −1}, corresponding to a solvent content of 44.8%.

  15. Crystallization and preliminary crystallographic studies of human kallikrein 7, a serine protease of the multigene kallikrein family

    Energy Technology Data Exchange (ETDEWEB)

    Fernández, Israel S. [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Ständker, Ludger [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Hannover Medical School, Center of Pharmacology, 30625 Hannover (Germany); Forssmann, Wolf-Georg [Hannover Medical School, Center of Pharmacology, 30625 Hannover (Germany); Giménez-Gallego, Guillermo; Romero, Antonio, E-mail: romero@cib.csic.es [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2007-08-01

    The cloning, expression, purification and crystallization of recombinant human kallikrein 7, directly synthesized in the active form in E. coli, is described. Diffraction data were collected to 2.8 Å resolution from native crystals. Human kallikreins are a group of serine proteases of high sequence homology whose genes are grouped as a single cluster at chromosome 19. Although the physiological roles of kallikreins are generally still unknown, members of the kallikrein family have been clearly implicated in pathological situations such as cancer and psoriasis. Human kallikrein 7 (hK7) has been shown to be involved in pathological keratinization, psoriasis and ovarian cancer. In order to gain insight into the molecular structure of this protein, hK7 was crystallized after recombinant production in its folded and active form using a periplasmic secretion vector in Escherichia coli. The crystals belonged to the rhombohedral space group H32 and diffracted to 2.8 Å. The phase problem was solved by molecular replacement using the mouse kallikrein-related protein neuropsin. Completion of the model and structure refinement are under way.

  16. Blood purification and hemo- perfusion

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The method of blood purification is a new overlapping frontierdiscipline which develops quickly in recent years. It helps overcoming many serious and complicated diseases, even including some incurable illnesses.

  17. Nanomechanical Water Purification Device Project

    Data.gov (United States)

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

  18. The SELEX Air Purification System

    Science.gov (United States)

    2010-01-07

    REPORT Final Report for the SELEX Air Purification System 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: A new air purification technology ( SELEX ) was...developed and demonstrated. The SELEX system utilizes an array of electrospray wick aerosol sources for particle ionization and an electrostatic...precipitator for particle collection. The particle ionization process does not produce ozone and the SELEX technology provides a unique combination of

  19. Expression, purification, and characterization of formaldehyde dehydrogenase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Zhang, Wangluo; Chen, Shuai; Liao, Yuanping; Wang, Dingli; Ding, Jianfeng; Wang, Yingming; Ran, Xiaoyuan; Lu, Daru; Zhu, Huaxing

    2013-12-01

    As a member of zinc-containing medium-chain alcohol dehydrogenase family, formaldehyde dehydrogenase (FDH) can oxidize toxic formaldehyde to less active formate with NAD(+) as a cofactor and exists in both prokaryotes and eukaryotes. Most FDHs are well known to be glutathione-dependent in the catalysis of formaldehyde oxidation, but the enzyme from Pseudomonas putida is an exception, which is independent of glutathione. To identify novel glutathione-independent FDHs from other bacterial strains and facilitate the corresponding structural and enzymatic studies, high-level soluble expression and efficient purification of these enzymes need to be achieved. Here, we present molecular cloning, expression, and purification of the FDH from Pseudomonas aeruginosa, which is a Gram-negative pathogenic bacterium causing opportunistic human infection. The FDH of P. aeruginosa shows high sequence identity (87.97%) with that of P. putida. Our results indicated that coexpression with molecular chaperones GroES, GroEL, and Tig has significantly attenuated inclusion body formation and improved the solubility of the recombinant FDH in Escherichiacoli cells. A purification protocol including three chromatographic steps was also established to isolate the recombinant FDH to homogeneity with a yield of ∼3.2 mg from 1L of cell culture. The recombinant P. aeruginosa FDH was properly folded and biologically functional, as demonstrated by the mass spectrometric, crystallographic, and enzymatic characterizations of the purified proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Placentation in cloned cattle

    DEFF Research Database (Denmark)

    Miglino, M A; Pereira, F T V; Visintin, J A

    2007-01-01

    To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies...... than one primary villus, as opposed to a single villus in non-cloned placentae. Scanning electron microscopy of blood vessel casts revealed that there was also more than one stem artery per villous tree and that the ramification of the vessels failed to form dense complexes of capillary loops...

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

    Directory of Open Access Journals (Sweden)

    Di-Hua Li

    2015-01-01

    Full Text Available 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.

  2. Why clone flies? Using cloned Drosophila to monitor epigenetic defects.

    Science.gov (United States)

    Haigh, Andrew J; Lloyd, Vett K

    2007-01-01

    Since the birth of the first cloned sheep in 1996, advances in nuclear transplantation have led to both the creation of genetically tailored stem cells and the generation of a number of cloned organisms. The list of cloned animals reared to adulthood currently includes the frog, sheep, mouse, cow, goat, pig, rabbit, cat, zebrafish, mule, horse, rat and dog. The addition of Drosophila to this elite bestiary of cloned animals has prompted the question - why clone flies? Organisms generated by nuclear transplantation suffer from a high rate of associated defects, and many of these defects appear to be related to aberrant genomic imprinting. Imprinted gene expression also appears to be compromised in Drosophila clones. Proper imprinted gene regulation relies on a suite of highly conserved chromatin-modifying genes first identified in Drosophila. Thus, Drosophila can potentially be used to study epigenetic dysfunction in cloned animals and to screen for genetic and epigenetic conditions that promote the production of healthy clones.

  3. Purification, crystallization and preliminary crystallographic analysis of AHP IX-bp, a zinc ion and pH-dependent coagulation factor IX binding protein from Agkistrodon halys Pallas venom.

    Science.gov (United States)

    Zang, Jianye; Teng, Maikun; Niu, Liwen

    2003-04-01

    A new coagulation factor IX binding protein, AHP IX-bp, has been purified from Agkistrodon halys Pallas venom and estimated to be an AB heterodimer of about 25 kDa consisting of two chains (an A chain of 15.5 kDa and a B chain of 15 kDa) linked by one or more disulfide bonds. The N-terminal sequence of AHP IX-bp has been determined and aligned with C-type lectin-like proteins. The protein has a high sequence similarity to some snake-venom C-type lectin-like proteins. AHP IX-bp binds to coagulation factor IX but not to coagulation factor X. Moreover, AHP IX-bp shows binding to coagulation factor IX in both zinc ion-dependent and pH-dependent manners. The affinity between AHP IX-bp and coagulation factor IX is higher under neutral or weakly alkaline conditions than under weakly acidic conditions. Single crystals of AHP IX-bp grown at pH 6.5 and 7.5 diffract X-rays to 2.0 and 1.8 A resolution, respectively. Both crystals are isomorphous and belong to the space group P1; only one AB heterodimer is present in the unit cell.

  4. Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Dziegiel, M; Borre, Mette; Jepsen, S

    1991-01-01

    A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P...... of the immunogenicity of a possible future P. falciparum vaccine utilizing epitopes from GLURP....

  5. Clip, connect, clone

    DEFF Research Database (Denmark)

    Fujima, Jun; Lunzer, Aran; Hornbæk, Kasper

    2010-01-01

    using three mechanisms: clipping of input and result elements from existing applications to form cells on a spreadsheet; connecting these cells using formulas, thus enabling result transfer between applications; and cloning cells so that multiple requests can be handled side by side. We demonstrate...

  6. The Cloning of America.

    Science.gov (United States)

    Dobson, Judith E.; Dobson, Russell L.

    1981-01-01

    Proposes that the U.S. school system purports to prize human variability, but many educators are engaged in activities that seek to homogenize students. Describes these activities, including diagnosis, labeling, ability grouping, and positive reinforcement. Presents suggestions for counselors to combat sources of cloning and self-validation. (RC)

  7. Asian Yellow Goat Cloned

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ It was released on August 24,2005 by Prof. CHEN Dayuan (Da-Yuan Chen) from the CAS Institute of Zoology that the first success in cloning the Asian Yellow Goat by nuclear transfer had recently been achieved in east China's Shandong Province.

  8. Separation process design for isolation and purification of natural products

    DEFF Research Database (Denmark)

    Malwade, Chandrakant R.

    selection of separation techniques and operating conditions. The key factor in designing separation processes with multiple unit operations is to determine the synergy between them which in turn demands molecular level understanding of process streams. Therefore, the methodology is fortified with process......, thereby providing process information crucial for determining synergistic effects between different unit operations. In this work, the formulated methodology has been used to isolate and purify artemisinin, an antimalarial drug, from dried leaves of the plant Artemisia annua. A process flow sheet...... is generated consisting of maceration, flash column chromatography and crystallization unit operations for extraction, partial purification and final purification of artemisinin, respectively. PAT framework is used extensively to characterize the process streams at molecular level and the generated process...

  9. Sequential cloning of chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1991-12-31

    A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  10. [Nuclear transfer and therapeutic cloning].

    Science.gov (United States)

    Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying

    2005-03-01

    Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

  11. The First Human Cloned Embryo.

    Science.gov (United States)

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  12. Animal Cloning and Food Safety

    Science.gov (United States)

    ... For Consumers Home For Consumers Consumer Updates Animal Cloning and Food Safety Share Tweet Linkedin Pin it ... evaluate the issue. back to top FDA Studies Cloning For more than five years, CVM scientists studied ...

  13. Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.

    Directory of Open Access Journals (Sweden)

    Manika Indrajit Singh

    Full Text Available We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3. Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR". We show the cloning, expression and purification of Green Fluorescent Protein (GFP as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3 with or without a hexa-histidine tag.

  14. Probabilistic Cloning and Quantum Computation

    Institute of Scientific and Technical Information of China (English)

    GAO Ting; YAN Feng-Li; WANG Zhi-Xi

    2004-01-01

    @@ We discuss the usefulness of quantum cloning and present examples of quantum computation tasks for which the cloning offers an advantage which cannot be matched by any approach that does not resort to quantum cloning.In these quantum computations, we need to distribute quantum information contained in the states about which we have some partial information. To perform quantum computations, we use a state-dependent probabilistic quantum cloning procedure to distribute quantum information in the middle of a quantum computation.

  15. Water Purification Systems

    Science.gov (United States)

    1994-01-01

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

  16. Neurotrophic factor - Characterization and partial purification

    Science.gov (United States)

    Popiela, H.; Ellis, S.

    1981-01-01

    Recent evidence suggests that neurotrophic activity is required for the normal proliferation and development of muscle cells. The present paper reports a study of the purification and characterization of a neurotrophic factor (NTF) from adult chicken ischiatic-peroneal nerves using two independent quantitative in vitro assay systems. The assays were performed by the measurement of the incorporation of tritiated thymidine or the sizes of single-cell clones by chick muscle cells grown in culture. The greatest amount of neutrotrophic activity is found to be extracted at a pH of 8; aqueous suspensions of the activity are stable to long-term storage at room temperature. The specific activity of the substance is doubled upon precipitation with ammonium sulfate or after gel filtration, and increase 4 to 5 fold after salt gradient elution from DEAE cellulose columns. The active fraction obtained after gel filtration and rechromatography on DEAE cellulose exhibits a 7 to 10-fold increase in specific activity. Electrophoresis of the most highly purified material yields a greatly concentrated band at around 80,000 daltons. Although NTF is purified almost 10-fold as indicated by the increase in specific activity, the maximum activity of the partially purified material is greatly reduced, possibly due to a requirement for a cofactor for the expression of maximum activity.

  17. Protocol for Initial Purification of Bacteriocin

    Science.gov (United States)

    2015-10-01

    lysate/extract preparation , column purification, and a desalting. The peptide was tracked throughout the process using a soft agar overlay activity... PREPARATION SOLUBLE EXTRACTS COLUMN CHROMATOGRAPHY BACTERIA PURIFICATION CHARACTERIZATION...3  6.1  Preparation of Target Bacteria

  18. Rapid purification of recombinant histones.

    Directory of Open Access Journals (Sweden)

    Henrike Klinker

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

  19. 古细菌-硫细菌蛋白AF1514的纯化及晶体生长研究%Study on Purification and Crystallization of Hypothetical Protein AF1514 from Archeoglobus fulgidus

    Institute of Scientific and Technical Information of China (English)

    帕孜来提·拜合提; 刘志杰; 阿不都拉·阿巴斯

    2008-01-01

    本研究将AF1514蛋白的重组基因转化到大肠杆菌(E.coli BL21)并通过在12℃条件下的诱导表达产生了大量的目的蛋白.用镍柱亲和层析和分子筛Superdex-75凝胶过滤层析的两步蛋白纯化方法进一步纯化蛋白AF1514后,获得纯度较高的蛋白(纯度>95%).纯化的母体蛋白和甲基化处理后的蛋白衍生物分别用悬滴汽相扩散法在16℃结晶.初步筛选所用的400多种结晶条件中,适合晶体生长的条件只有3种,其中最佳晶体生长条件溶液的成分包含0.1mol/L醋酸钠,pH 5.0,0.1mol/L氯化钠和8%~14%(W/V)2-甲基2,4-戊二醇.蛋白晶体属于四方晶系,初步X-射线衍射分辨率为2.09A°.%Archeoglobus fulgidus DSM 4304 genome was transformed and overproduced in Escherichia coli(E.coli BL21)at 12℃and the protein was purified to 95%purity by Ni2+-affinity colunm and Superdex-75 gel filtration.Crystals ative and methylated proteins were obtained by the hanging-drop vapour-diffusion method at 16℃under three conditions among the wide range of screened conditions(>400),but principally from the solution containing 0.1mol/L sodium acetate pH 5.0,0.1mol/L sodium chloride,and 8%~14%(W/V)2-methyl-2,4-dipentaneol (MPD).The crystal is tetragonal in shape and diffraction data to 2.09A° are detected by X-ray detector.

  20. Introduction to protein crystallization.

    Science.gov (United States)

    McPherson, Alexander; Gavira, Jose A

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

  1. Purification of Water by Aquatic Plants

    OpenAIRE

    Morimitsu, Katsuhito; Kawahigashi, Tatsuo

    2013-01-01

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

  2. Purification, partial characterization, crystallization and structural determination of AHP-LAAO, a novel L-amino-acid oxidase with cell apoptosis-inducing activity from Agkistrodon halys pallas venom.

    Science.gov (United States)

    Zhang, Hongmin; Teng, Maikun; Niu, Liwen; Wang, Yubao; Wang, Yuzhen; Liu, Qun; Huang, Qingqiu; Hao, Quan; Dong, Yuhui; Liu, Peng

    2004-05-01

    A snake-venom protein named AHP-LAAO has been purified from Agkistrodon halys pallas venom using four-stage chromatography. AHP-LAAO is a novel member of the snake-venom L-amino-acid oxidase family. Its amino-acid sequence shows high homology to other members of this family. For L-leucine, the values of k(cat) and K(M) are 31.1 s(-1) and 0.25 mM, respectively. The molecular weight of AHP-LAAO is about 60.7 kDa as determined by MALDI-TOF mass spectrometry. AHP-LAAO can also induce apoptosis of cultured Hela cells. Two sets of diffraction data with similar resolution limits (about 2.5 A) were collected independently at MacCHESS (Cornell High Energy Synchrotron Source, USA) and IHEP (Institute of High Energy Physics, Beijing, China). The crystals belong to space group I2(1)3, with unit-cell parameter a = 169.31 A, corresponding to one molecule in the asymmetric unit and a volume-to-weight ratio of 3.33 A(3) Da(-1). The final structural model is similar to that of L-amino-acid oxidase from Calloselasma rhodostoma venom.

  3. Entering the Clone Age

    Institute of Scientific and Technical Information of China (English)

    1995-01-01

    Suppose you make your parents so happy,they decide to have another baby just like you.It might be flattering,but how would you feel about having a little brother or sister who is also your twin? A laboratory experiment conducted last fall suggests it may someday be possible.For the first time ever,scientists made exact copies, or clones, of a human embryo.

  4. RELIGION AND PURIFICATION OF SOUL

    Directory of Open Access Journals (Sweden)

    Azam Khodashenas Pelko

    2010-11-01

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

  5. Sequential cloning of chromosomes

    Science.gov (United States)

    Lacks, S.A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes. 9 figs.

  6. Cloning-free CRISPR

    Directory of Open Access Journals (Sweden)

    Mandana Arbab

    2015-11-01

    Full Text Available We present self-cloning CRISPR/Cas9 (scCRISPR, a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.

  7. Secure the Clones

    CERN Document Server

    Jensen, Thomas; Pichardie, David

    2012-01-01

    Exchanging mutable data objects with untrusted code is a delicate matter because of the risk of creating a data space that is accessible by an attacker. Consequently, secure programming guidelines for Java stress the importance of using defensive copying before accepting or handing out references to an internal mutable object. However, implementation of a copy method (like clone()) is entirely left to the programmer. It may not provide a sufficiently deep copy of an object and is subject to overriding by a malicious sub-class. Currently no language-based mechanism supports secure object cloning. This paper proposes a type-based annotation system for defining modular copy policies for class-based object-oriented programs. A copy policy specifies the maximally allowed sharing between an object and its clone. We present a static enforcement mechanism that will guarantee that all classes fulfil their copy policy, even in the presence of overriding of copy methods, and establish the semantic correctness of the ove...

  8. Ethical issues in livestock cloning.

    Science.gov (United States)

    Thompson, P B

    1999-01-01

    Although cloning may eventually become an important technology for livestock production, four ethical issues must be addressed before the practice becomes widespread. First, researchers must establish that the procedure is not detrimental to the health or well-being of affected animals. Second, animal research institutions should evaluate the net social benefits to livestock producers by weighing the benefits to producers against the opportunity cost of research capacity lost to biomedical projects. Third, scientists should consider the indirect effects of cloning research on the larger ethical issues surrounding human cloning. Finally, the market structure for products of cloned animals should protect individual choice, and should recognize that many individuals find the prospect of cloning (or consuming cloned animals) repugnant. Analysis of these four issues is complicated by spurious arguments alleging that cloning will have a negative impact on environment and genetic diversity.

  9. A new method to customize protein expression vectors for fast, efficient and background free parallel cloning

    OpenAIRE

    Scholz, J; Besir, H.; Strasser, C.; Suppmann, S.

    2013-01-01

    Background: Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector. Results: Here we describe a method to tailor selected expression vectors for para...

  10. Hydrogen purification by periodic adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Barg, Christian; Secchi, Argimiro R.; Trierweiler, Jorge O. [Rio Grande do Sul Univ., Porto Alegre, RS (Brazil). Dept. de Engenharia Quimica]. E-mail: cbarg@enq.ufrgs.br; arge@enq.ufrgs.br; jorge@enq.ufrgs.br

    2000-07-01

    The periodic adsorption processes have been widely used for industrial applications, mainly because it spends less energy than the usual gas separation processes, like the cryogenic distillation. The largest commercial application of periodic adsorption processes is the pressure swing adsorption (PSA) applied to hydrogen purification. Although its wide use in the chemical and petrochemical industry, there are no reports in the open literature about complete modeling studies of a complex commercial unit, with multiple adsorbents and multiple beds and several feed components. This study has as objective the modeling, optimization and dynamical analysis of an industrial PSA unit for hydrogen purification. (author)

  11. Cloning and Expression of Ontak Immunotoxin Using Intein Tag

    Directory of Open Access Journals (Sweden)

    SA Moosavizadeh

    2016-06-01

    Full Text Available Introduction: Inteins (INT are internal parts of a number of proteins in yeast and some other unicellular eukaryotes, which can be separated from the immature protein during protein splicing process. After identifying the mechanism of intein action, applications of these sequences are be considered in the single- step purification of recombinant proteins and different intein tags were developed. The most important advantage of using intein tags in purification of recombinant proteins than other affinity tags is no requirement of expensive protease enzymes and following additional steps to remove protease that make intein tags economically are considered more important. In the present study, denileukin diftitox immunotoxin (brand name Ontak, be fused with an intein tag and it was inserted in pTXB1 plasmid. Methods: In this study, with respect to multiple cloning sites (MCS of pTXB1, specific primers were designed. Polymerase Chain Reaction (PCR was performed and encoding sequence of ONTAK was cloned using restriction sites of NdeI and SapI. Recombinant vector (PTX-IDZ was transformed into E. coli strain ER2566 and expression of gene was studied. Results: The accuracy of recombinant construct was confirmed by PCR and enzymatic digestion. The produced recombinant proteins were confirmed by SDS-PAGE and Western blotting. Conclusion: Restriction site of SapI guarantees no additional residues incorporate in primary protein sequence. Also, the expression of this construct was analyzed in compare with fused protein to poly-His tag. According to the appropriate expression of fused protein in both constructs it was expected that one step- purification of considered drug protein will be success in the following steps.

  12. Novel cloning vectors for Bacillus thuringiensis.

    Science.gov (United States)

    Baum, J A; Coyle, D M; Gilbert, M P; Jany, C S; Gawron-Burke, C

    1990-11-01

    Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.

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

    Science.gov (United States)

    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.

  14. Influence of Impurities on the Radiation Response of the TlBr Semiconductor Crystal

    Directory of Open Access Journals (Sweden)

    Robinson Alves dos Santos

    2017-01-01

    Full Text Available Two commercially available TlBr salts were used as the raw material for crystal growths to be used as radiation detectors. Previously, TlBr salts were purified once, twice, and three times by the repeated Bridgman method. The purification efficiency was evaluated by inductively coupled plasma mass spectroscopy (ICP-MS, after each purification process. A compartmental model was proposed to fit the impurity concentration as a function of the repetition number of the Bridgman growths, as well as determine the segregation coefficients of impurities in the crystals. The crystalline structure, the stoichiometry, and the surface morphology of the crystals were evaluated, systematically, for the crystals grown with different purification numbers. To evaluate the crystal as a radiation semiconductor detector, measurements of its resistivity and gamma-ray spectroscopy were carried out, using 241Am and 133Ba sources. A significant improvement of the radiation response was observed in function of the crystal purity.

  15. 布鲁氏菌外膜脂蛋白OMP10基因的克隆表达、蛋白纯化及免疫原性研究%Cloning, Expression, Purification and Immunogenicity Test of Brucella Lipoprotein OMP10

    Institute of Scientific and Technical Information of China (English)

    马长胜; 丁家波; 杨宏军; 何洪彬; 宋玲玲; 侯佩莉; 仲跻峰; 谢之景

    2012-01-01

    根据GenBank中的牛型布鲁氏杆菌OMP10序列,设计一对引物,以布鲁氏菌疫苗株S2全基因组DNA为模板扩增OMP10基因,将目的基因克隆人pEASY -T3,经测序正确后,与载体pET - 32a(+)连接,构建重组表达质粒pET - 32a/OMP10,转化E.coli BL21( DE3),用IPTG诱导表达融合蛋白pET - 32a/OMP10,经SDS - PAGE及Western - blot分析鉴定后,采用Ni- NTA Spin Kit纯化目的蛋白,并免疫小鼠.结果表明,成功构建了含OMP10的表达载体,经多次对表达条件的优化,获得了纯度较高的目的蛋白,为进一步动物免疫试验及研究其免疫原性和抗感染保护作用奠定了基础.%To construct a prokaryotic expression vector for Brucella OMP10, as well as express and purify the recombinant protein, Brucella S2 genome sequence was extracted and used as temple for polymerase chain reaction to amplify OMPIO gene. Then the PCR product was cloned into the pEASY - T3 vector and subsequently sequenced. Then the gene was cloned into the expression vector pET - 32a ( + ) to construct the recombinant plasmid pET -32a/OMP10. The fused protein pET -32a/OMP10 was expressed by IPTG induction and examined by SDS - PAGE and Western - blot, then purified by Ni - NTA Spin Kit. In this study, Brucella OMPIO was successfully cloned, the expression condition was optimized, the target protein was strongly expressed, and highly purified protein was obtained. This study laid a foundation for further research of animal examination, immunogenicity and protection of brucellosis.

  16. Ethical issues in animal cloning.

    Science.gov (United States)

    Fiester, Autumn

    2005-01-01

    The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.

  17. Purification and properties of dialkylfluorophosphatase

    NARCIS (Netherlands)

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

    1957-01-01

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

  18. Quantum entanglement purification in cavities

    CERN Document Server

    Romero, J L; Saavedra, C; Retamal, J C

    2002-01-01

    A physical implementation of an entanglement purification protocol is studied using a cavity quantum electrodynamic based proposal, where, the quantum information is stored in quantum field sates inside cavities. Also a procedure is given for quantifying the degree of entanglement between quantum fields. (Author)

  19. Bioinspired Materials for Water Purification

    Directory of Open Access Journals (Sweden)

    Alfredo Gonzalez-Perez

    2016-06-01

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

  20. [Immobilized microorganisms and water purification].

    Science.gov (United States)

    Mogilevich, N F

    1995-01-01

    Advantages and disadvantages of cells of aerobic microorganisms immobilized by the type of adhesion and incorporation into the gel beads, the amount of retained biomass, limitations of diffusion of oxygen and nutrients, viability, morphology, biochemical properties are described. Immobilized biocatalysts are discussed in the aspect of their use in purification of sewage waters.

  1. Purification and concentration of alphavirus.

    Science.gov (United States)

    Lundstrom, Kenneth

    2012-07-01

    The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Membrane proteins (which are generally difficult to express at high levels in recombinant systems) have generated high yields and facilitate applications in structural biology. Alphaviruses have also been applied in vaccine development and gene therapy. Generally, purification or concentration of alphaviruses is not necessary. However, for instance, the medium derived from baby hamster kidney cells is toxic to primary neurons in culture. Including a purification step substantially improves the survival of the transduced neurons. Viral concentration and purification may also be advantageous for in vivo studies in animal models and are mandatory for clinical applications. This protocol describes three methods for purification and concentration of alphavirus.

  2. Biobased monoliths for adenovirus purification.

    Science.gov (United States)

    Fernandes, Cláudia S M; Gonçalves, Bianca; Sousa, Margarida; Martins, Duarte L; Barroso, Telma; Pina, Ana Sofia; Peixoto, Cristina; Aguiar-Ricardo, Ana; Roque, A Cecília A

    2015-04-01

    Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (-20 °C and -80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at -80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

  3. To clone or not to clone--whither the law?

    Science.gov (United States)

    Lupton, M L

    1999-01-01

    The cloning of Dolly the lamb from adult cells by scientists at the Roslin Laboratories near Edinburgh in February 1997 has startled the world because it now opens the way to clone adult human beings. The reaction to Ian Wilmut's breakthrough has been instant and largely negative. Bills were rushed into both the US Senate and House of Representatives aimed at banning the cloning of human beings. Human cloning is premature at this stage, but there are many positive spin-offs of cloning in the field of genetic engineering, such as the production of human proteins such as blood clotting factors which aid in healing wounds. Progress by means of cloning can also be made into devising a cure for Parkinson's Disease amongst others. No lesser ethicist than John C. Fletcher of the University of Virginia foresees circumstances in which human cloning is acceptable e.g. to enable a couple to replace a dying child, to enable a couple, one of whom is infertile, to clone a child from either partner. Extensive regulation of cloning by the law is inevitable but, in doing so, the legislation should be careful not to outlaw research in this area which could be beneficial to mankind.

  4. Lessons learned from cloning dogs.

    Science.gov (United States)

    Kim, M J; Oh, H J; Kim, G A; Park, J E; Park, E J; Jang, G; Ra, J C; Kang, S K; Lee, B C

    2012-08-01

    The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals.

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

    Directory of Open Access Journals (Sweden)

    Alex Jose José de Melo Silva

    2016-12-01

    Full Text Available This work was performed as an integrated practical of a Biomedical Science undergraduate course of Biochemistry subject, in order to demonstrate used techniques to purify of Green Fluorescent Protein (GFP. To perform the experiments the main methodology applied was the by immobilized metal affinity chromatography (IMAC.  The open reading frame for enhanced GFP was sub-cloned into the pQE30 expression vector. The subsequent production of protein tagged N-terminally with hexahistidine, facilitated its purification by IMAC.  An approximate 3-fold purification of GFP was achieved. Thus, the students who completed the course gained significant experience related to fundamental techniques in molecular cloning and a sound basis in the principles of recombinant protein expression and purification.

  6. Molecular Cloning of Adenosinediphosphoribosyl Transferase.

    Science.gov (United States)

    1987-09-08

    ACCESSION NO.D,. 03261102F 2312 A~5 11. TITLE (include Securqt Classification) 0 Molecular Cloning of Adenosinediphosphoribosyl Transferase 12. PERSONAL...I’:- AFOSR.Tlt. 8 7 - 0 9 8,2 0IL * pi AFOSR- 85 -0377 PROGRESS REPORT Molecular Cloning of Adenosinediphosphoribosyl Transferase 5." Period of...Pharmacology and the Cardiovascular Research Institute September 8, 1987 .’, 5.’- "’S ". -f, AFOSR - 85 -0377 PROGRESS REPORT Molecular Cloning of

  7. An in silico DNA cloning experiment for the biochemistry laboratory.

    Science.gov (United States)

    Elkins, Kelly M

    2011-01-01

    This laboratory exercise introduces students to concepts in recombinant DNA technology while accommodating a major semester project in protein purification, structure, and function in a biochemistry laboratory for junior- and senior-level undergraduate students. It is also suitable for forensic science courses focused in DNA biology and advanced high school biology classes. Students begin by examining a plasmid map with the goal of identifying which restriction enzymes may be used to clone a piece of foreign DNA containing a gene of interest into the vector. From the National Center for Biotechnology Initiative website, students are instructed to retrieve a protein sequence and use Expasy's Reverse Translate program to reverse translate the protein to cDNA. Students then use Integrated DNA Technologies' OligoAnalyzer to predict the complementary DNA strand and obtain DNA recognition sequences for the desired restriction enzymes from New England Biolabs' website. Students add the appropriate DNA restriction sequences to the double-stranded foreign DNA for cloning into the plasmid and infecting Escherichia coli cells. Students are introduced to computational biology tools, molecular biology terminology and the process of DNA cloning in this valuable single session, in silico experiment. This project develops students' understanding of the cloning process as a whole and contrasts with other laboratory and internship experiences in which the students may be involved in only a piece of the cloning process/techniques. Students interested in pursuing postgraduate study and research or employment in an academic biochemistry or molecular biology laboratory or industry will benefit most from this experience.

  8. Human cloning and child welfare.

    Science.gov (United States)

    Burley, J; Harris, J

    1999-01-01

    In this paper we discuss an objection to human cloning which appeals to the welfare of the child. This objection varies according to the sort of harm it is expected the clone will suffer. The three formulations of it that we will consider are: 1. Clones will be harmed by the fearful or prejudicial attitudes people may have about or towards them (H1); 2. Clones will be harmed by the demands and expectations of parents or genotype donors (H2); 3. Clones will be harmed by their own awareness of their origins, for example the knowledge that the genetic donor is a stranger (H3). We will show why these three versions of the child welfare objection do not necessarily supply compelling reasons to ban human reproductive cloning. The claim that we will develop and defend in the course of our discussion is that even if it is the case that a cloned child will suffer harms of the type H1-H3, it is none the less permissible to conceive by cloning so long as these cloning-induced welfare deficits are not such as to blight the existence of the resultant child, whoever this may be. PMID:10226914

  9. Crystals in crystals

    DEFF Research Database (Denmark)

    Christensen, Claus H.; Schmidt, I.; Carlsson, A.;

    2005-01-01

    A major factor governing the performance of catalytically active particles supported on a zeolite carrier is the degree of dispersion. It is shown that the introduction of noncrystallographic mesopores into zeolite single crystals (silicalite-1, ZSM-5) may increase the degree of particle dispersion...... of the zeolite particles, particularly after thermal treatment. When using mesoporous zeolites, the particles were evenly distributed throughout the mesopore system of the zeolitic support, even after calcination, leading to nanocrystals within mesoporous zeolite single crystals....

  10. Production and purification of active snowdrop lectin in Escherichia coli.

    Science.gov (United States)

    Longstaff, M; Powell, K S; Gatehouse, J A; Raemaekers, R; Newell, C A; Hamilton, W D

    1998-02-15

    Recombinant snowdrop lectin was produced in Escherichia coli from a cDNA clone encoding mature Galanthus nivalis agglutinin. After induction with isopropylthio-beta-D-galactoside, inclusion bodies from E. coli were solubilised and the G. nivalis agglutinin purified by metal-affinity chromatography using a carboxy-terminal hexahistidine tag. The protein was refolded on the metal-affinity column prior to elution. After purification, the recombinant G. nivalis agglutinin agglutinated rabbit erythrocytes to a dilution similar to that determined for 'native' lectin purified from snowdrop, and showed similar specific binding to mannose. The toxicity of the recombinant G. nivalis agglutinin towards rice brown planthopper (Nilaparvata lugens) was shown to be similar to that of 'native' G. nivalis agglutinin when incorporated into an artificial diet. The recombinant G. nivalis agglutinin is thus functionally similar to 'native' snowdrop lectin.

  11. Efficient expression and purification of biologically active human cystatin proteins.

    Science.gov (United States)

    Chauhan, Sakshi; Tomar, Raghuvir S

    2016-02-01

    Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

  12. A novel purification process for dodecanedioic acid by molecular distillation

    Institute of Scientific and Technical Information of China (English)

    Jiang Yu; Xigang Yuan; Aiwu Zeng

    2015-01-01

    A novel purification process is involved to obtain the high purity [N 99%(by mass)] dodecanedioic acid (DC12). It involves a re-crystal ization followed by molecular distil ation from the crude product. The objective of this study is to investigate general conditions, feed rate, distil ing temperature and vacuum, necessary for centrifugal distil-lation of DC12. Under the optimum conditions, distilling temperature 180 °C, pressure 30 Pa and feed flow rate 700 ml·h−1, the purity of DC12 in the residence reached 97.55%with a yield of 53.18%by the analysis of gas chromatography. Multiple-pass distillation made a considerable contribution by improving the purity to 99.22%. Additionally, the effect of pretreatment (re-crystallization) on distillation process was revealed through a series of comparative experiments.

  13. Membrane adsorbers as purification tools for monoclonal antibody purification.

    Science.gov (United States)

    Boi, Cristiana

    2007-03-15

    Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.

  14. 直接分子克隆法构建重组腺病毒AdHu5-mfsp27的研究%Direct molecular cloning to construct recombinant AdHu5-mfsp27 and its amplification and purification

    Institute of Scientific and Technical Information of China (English)

    王群; 贾蓓; 魏晓宇; 黄文祥; 周向阳

    2012-01-01

    Objective The aim of this study was to construct recombinant AdHu5-040-mfsp27 (cds) using direct molecular cloning and obtain viruses with a high titer and level of purity for laboratory and clinical trials. Methods The target gene mfsp27 was cloned into pShuttle and located between two rare restriction enzymes (REs) (I-ceuI and PI-sceI). After digestion, gel ligation, and transformation, recombinants were selected and confirmed by specific RE cutting and sequencing. The candidate recombinant plasmid was then linearized and transfected into HEK293 cells. After infection and expansion, the crude virus stock was collected and purified by modified cesium chloride gradient centrifugation. The virus multiplicity of infection (MOD was measured by series dilution of the stock. Results Recombinant AdHu5-040-fsp27 (cds) was successfully constructed. The titer (O. D. measurement) was as high as to 1 × 1013 VP/ ml and MOI was 1. 44 × 1011. The ratio of the two values was 69. 44. The recombinant virus had potent ability to infect HEK293 cells. Conclusion Direct molecular cloning to construct recombinant Ad Hu5-040-fsp27 (cds) is better than homologous recombination because it takes less time and it provides highly positive clones with fewer mutations of target genes. Modified cesium chloride gradient centrifugation combined with a liquid chromatography column and biogels is a simple and efficient way to obtain purified viruses in high tilers and with a high level of activity. This provides an experimental basis for the study of gene function and the industrial production of recombinant adenovirus.%目的 采用直接分子克隆体外连接的方法构建重组腺病毒AdHu5-040-mfsp27(cds),高效高质量扩增和纯化重组腺病毒以满足实验及临床研究的需求. 方法 将mfsp27克隆至穿梭质粒pSh CMV intron eGFP两个稀有限制性内切酶之间(I-ceuI和PI-sceI)(与腺病毒骨架质粒相同),回收酶切片段后连接,用合适的感受态细胞转

  15. [The discrete horror of cloning].

    Science.gov (United States)

    Guibourg, Ricardo A

    2009-01-01

    The author raises the topic of cloning after the decision of the Argentine government, which concerned for the "dignity of the human person", passed a decree of need and urgency, No. 200/97 (Annex), prohibiting cloning experiments with human beings. Therefore, considering that the topic is so terribly urgent and necessary, the author feels it is timely to consider it.

  16. CATO: The Clone Alignment Tool.

    Directory of Open Access Journals (Sweden)

    Peter V Henstock

    Full Text Available High-throughput cloning efforts produce large numbers of sequences that need to be aligned, edited, compared with reference sequences, and organized as files and selected clones. Different pieces of software are typically required to perform each of these tasks. We have designed a single piece of software, CATO, the Clone Alignment Tool, that allows a user to align, evaluate, edit, and select clone sequences based on comparisons to reference sequences. The input and output are designed to be compatible with standard data formats, and thus suitable for integration into a clone processing pipeline. CATO provides both sequence alignment and visualizations to facilitate the analysis of cloning experiments. The alignment algorithm matches each of the relevant candidate sequences against each reference sequence. The visualization portion displays three levels of matching: 1 a top-level summary of the top candidate sequences aligned to each reference sequence, 2 a focused alignment view with the nucleotides of matched sequences displayed against one reference sequence, and 3 a pair-wise alignment of a single reference and candidate sequence pair. Users can select the minimum matching criteria for valid clones, edit or swap reference sequences, and export the results to a summary file as part of the high-throughput cloning workflow.

  17. CATO: The Clone Alignment Tool.

    Science.gov (United States)

    Henstock, Peter V; LaPan, Peter

    2016-01-01

    High-throughput cloning efforts produce large numbers of sequences that need to be aligned, edited, compared with reference sequences, and organized as files and selected clones. Different pieces of software are typically required to perform each of these tasks. We have designed a single piece of software, CATO, the Clone Alignment Tool, that allows a user to align, evaluate, edit, and select clone sequences based on comparisons to reference sequences. The input and output are designed to be compatible with standard data formats, and thus suitable for integration into a clone processing pipeline. CATO provides both sequence alignment and visualizations to facilitate the analysis of cloning experiments. The alignment algorithm matches each of the relevant candidate sequences against each reference sequence. The visualization portion displays three levels of matching: 1) a top-level summary of the top candidate sequences aligned to each reference sequence, 2) a focused alignment view with the nucleotides of matched sequences displayed against one reference sequence, and 3) a pair-wise alignment of a single reference and candidate sequence pair. Users can select the minimum matching criteria for valid clones, edit or swap reference sequences, and export the results to a summary file as part of the high-throughput cloning workflow.

  18. [Scientific ethics of human cloning].

    Science.gov (United States)

    Valenzuela, Carlos Y

    2005-01-01

    True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.

  19. Quantum probabilistically cloning and computation

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this article we make a review on the usefulness of probabilistically cloning and present examples of quantum computation tasks for which quantum cloning offers an advantage which cannot be matched by any approach that does not resort to it.In these quantum computations,one needs to distribute quantum information contained in states about which we have some partial information.To perform quantum computations,one uses state-dependent probabilistic quantum cloning procedure to distribute quantum information in the middle of a quantum computation.And we discuss the achievable efficiencies and the efficient quantum logic network for probabilistic cloning the quantum states used in implementing quantum computation tasks for which cloning provides enhancement in performance.

  20. Purification of Tetrahymena cytoskeletal proteins.

    Science.gov (United States)

    Honts, Jerry E

    2012-01-01

    Like all eukaryotic cells, Tetrahymena thermophila contains a rich array of cytoskeletal proteins, some familiar and some novel. A detailed analysis of the structure, function, and interactions of these proteins requires procedures for purifying the individual protein components. Procedures for the purification of actin and tubulin from Tetrahymena are reviewed, followed by a description of a procedure that yields proteins from the epiplasmic layer and associated structures, including the tetrins. Finally, the challenges and opportunities for future advances are assessed.