The Experts below are selected from a list of 5331 Experts worldwide ranked by ideXlab platform

William B. Whitman - One of the best experts on this subject based on the ideXlab platform.

  • isolation of acetate auxotrophs of the methane producing archaeon methanococcus maripaludis by random insertional mutagenesis
    Genetics, 1999
    Co-Authors: William B. Whitman
    Abstract:

    To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide Plasmid bearing the puromycin-resistance cassette pac. These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth. One mutant, JJ104, was a leaky acetate auxotroph. A Plasmid, pWDK104, was recovered from this mutant by electroporation of a Plasmid Preparation into Escherichia coli. Transformation of wild-type M. maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype. pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M. jannaschii. Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions. A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both. Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin. This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism. Plasmids rescued from other mutants contained portions of ORFs denoted in M. jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1).

  • isolation of acetate auxotrophs of the methane producing archaeon methanococcus maripaludis by random insertional mutagenesis
    Genetics, 1999
    Co-Authors: William B. Whitman
    Abstract:

    To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide Plasmid bearing the puromycin-resistance cassette pac. These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth. One mutant, JJ104, was a leaky acetate auxotroph. A Plasmid, pWDK104, was recovered from this mutant by electroporation of a Plasmid Preparation into Escherichia coli. Transformation of wild-type M. maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype. pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M. jannaschii. Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions. A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both. Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin. This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism. Plasmids rescued from other mutants contained portions of ORFs denoted in M. jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1).

Duarte M F Prazeres - One of the best experts on this subject based on the ideXlab platform.

  • purification of a cystic fibrosis Plasmid vector for gene therapy using hydrophobic interaction chromatography
    Biotechnology and Bioengineering, 2000
    Co-Authors: Maria Margarida Diogo, Joao A Queiroz, Gabriel A Monteiro, S A M Martins, Guilherme N M Ferreira, Duarte M F Prazeres
    Abstract:

    The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of Plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis Plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded Plasmid DNA. RNA, denatured genomic and Plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled Plasmid, were retained and separated from nonbinding Plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded Plasmid was recovered after HIC. RNA and denatured Plasmid in the final Plasmid Preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/μg pDNA and 0.048 EU/μg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final Preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 576–583, 2000.

  • purification of a cystic fibrosis Plasmid vector for gene therapy using hydrophobic interaction chromatography
    Biotechnology and Bioengineering, 2000
    Co-Authors: Maria Margarida Diogo, Joao A Queiroz, Gabriel A Monteiro, S A M Martins, Guilherme N M Ferreira, Duarte M F Prazeres
    Abstract:

    The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of Plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis Plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded Plasmid DNA. RNA, denatured genomic and Plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled Plasmid, were retained and separated from nonbinding Plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded Plasmid was recovered after HIC. RNA and denatured Plasmid in the final Plasmid Preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final Preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.

Gabriel A Monteiro - One of the best experts on this subject based on the ideXlab platform.

  • purification of a cystic fibrosis Plasmid vector for gene therapy using hydrophobic interaction chromatography
    Biotechnology and Bioengineering, 2000
    Co-Authors: Maria Margarida Diogo, Joao A Queiroz, Gabriel A Monteiro, S A M Martins, Guilherme N M Ferreira, Duarte M F Prazeres
    Abstract:

    The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of Plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis Plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded Plasmid DNA. RNA, denatured genomic and Plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled Plasmid, were retained and separated from nonbinding Plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded Plasmid was recovered after HIC. RNA and denatured Plasmid in the final Plasmid Preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/μg pDNA and 0.048 EU/μg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final Preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 576–583, 2000.

  • purification of a cystic fibrosis Plasmid vector for gene therapy using hydrophobic interaction chromatography
    Biotechnology and Bioengineering, 2000
    Co-Authors: Maria Margarida Diogo, Joao A Queiroz, Gabriel A Monteiro, S A M Martins, Guilherme N M Ferreira, Duarte M F Prazeres
    Abstract:

    The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of Plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis Plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded Plasmid DNA. RNA, denatured genomic and Plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled Plasmid, were retained and separated from nonbinding Plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded Plasmid was recovered after HIC. RNA and denatured Plasmid in the final Plasmid Preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final Preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.

S A M Martins - One of the best experts on this subject based on the ideXlab platform.

  • purification of a cystic fibrosis Plasmid vector for gene therapy using hydrophobic interaction chromatography
    Biotechnology and Bioengineering, 2000
    Co-Authors: Maria Margarida Diogo, Joao A Queiroz, Gabriel A Monteiro, S A M Martins, Guilherme N M Ferreira, Duarte M F Prazeres
    Abstract:

    The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of Plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis Plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded Plasmid DNA. RNA, denatured genomic and Plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled Plasmid, were retained and separated from nonbinding Plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded Plasmid was recovered after HIC. RNA and denatured Plasmid in the final Plasmid Preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/μg pDNA and 0.048 EU/μg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final Preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 576–583, 2000.

  • purification of a cystic fibrosis Plasmid vector for gene therapy using hydrophobic interaction chromatography
    Biotechnology and Bioengineering, 2000
    Co-Authors: Maria Margarida Diogo, Joao A Queiroz, Gabriel A Monteiro, S A M Martins, Guilherme N M Ferreira, Duarte M F Prazeres
    Abstract:

    The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of Plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis Plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded Plasmid DNA. RNA, denatured genomic and Plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled Plasmid, were retained and separated from nonbinding Plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded Plasmid was recovered after HIC. RNA and denatured Plasmid in the final Plasmid Preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final Preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.

Maria Margarida Diogo - One of the best experts on this subject based on the ideXlab platform.

  • purification of a cystic fibrosis Plasmid vector for gene therapy using hydrophobic interaction chromatography
    Biotechnology and Bioengineering, 2000
    Co-Authors: Maria Margarida Diogo, Joao A Queiroz, Gabriel A Monteiro, S A M Martins, Guilherme N M Ferreira, Duarte M F Prazeres
    Abstract:

    The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of Plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis Plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded Plasmid DNA. RNA, denatured genomic and Plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled Plasmid, were retained and separated from nonbinding Plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded Plasmid was recovered after HIC. RNA and denatured Plasmid in the final Plasmid Preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/μg pDNA and 0.048 EU/μg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final Preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 576–583, 2000.

  • purification of a cystic fibrosis Plasmid vector for gene therapy using hydrophobic interaction chromatography
    Biotechnology and Bioengineering, 2000
    Co-Authors: Maria Margarida Diogo, Joao A Queiroz, Gabriel A Monteiro, S A M Martins, Guilherme N M Ferreira, Duarte M F Prazeres
    Abstract:

    The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of Plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis Plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded Plasmid DNA. RNA, denatured genomic and Plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled Plasmid, were retained and separated from nonbinding Plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded Plasmid was recovered after HIC. RNA and denatured Plasmid in the final Plasmid Preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final Preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.