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  • American Society for Microbiology  (2)
  • Biology  (2)
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  • American Society for Microbiology  (2)
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  • Biology  (2)
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  • 1
    Online Resource
    Online Resource
    Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production
    American Society for Microbiology ; 2012
    In:  Applied and Environmental Microbiology Vol. 78, No. 23 ( 2012-12), p. 8454-8462
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 78, No. 23 ( 2012-12), p. 8454-8462
    Abstract: Pseudomonas fluorescens , a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production. P. fluorescens possesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins in P. fluorescens are attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from the P. fluorescens genome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes of P. fluorescens SIK W1 were deleted using the targeted gene knockout method. Deletion mutant P. fluorescens Δ tliA Δ prtA secreted fusion proteins without TliA or protein degradation. Using wild-type P. fluorescens as an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with the P. fluorescens Δ tliA Δ prtA double mutant irrespective of growth conditions. By homologous expression of tliA and the ABC transporter in a plasmid, TliA secreted from P. fluorescens Δ prtA and P. fluorescens Δ tliA Δ prtA cells was found to be intact, whereas that secreted from the wild-type P. fluorescens and P. fluorescens Δ tliA cells was found to be hydrolyzed. Our results demonstrate that the P. fluorescens Δ tliA Δ prtA deletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02476-12
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 2
    Online Resource
    Online Resource
    Use of Cell Culture-PCR Assay Based on Combination of A549 and BGMK Cell Lines and Molecular Identification as a Tool To Monitor Infectious Adenoviruses and Enteroviruses in River Water
    American Society for Microbiology ; 2004
    In:  Applied and Environmental Microbiology Vol. 70, No. 11 ( 2004-11), p. 6695-6705
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 70, No. 11 ( 2004-11), p. 6695-6705
    Abstract: Viral contamination in environmental samples can be underestimated because a single cell line might reproduce only some enteric viruses and some enteric viruses do not exhibit apparent cytopathic effects in cell culture. To overcome this problem, we evaluated a cell culture-PCR assay based on a combination of A549 and Buffalo green monkey kidney (BGMK) cell lines as a tool to monitor infectious adenoviruses and enteroviruses in river water. Water samples were collected 10 times at each of four rivers located in Gyeonggi Province, South Korea, and then cultured on group 1 cells (BGMK cells alone) and group 2 cells (BGMK and A549 cells). Reverse transcription and multiplex PCR were performed, followed by phylogenetic analysis of the amplicons. Thirty (75.0%) of the 40 samples were positive for viruses based on cell culture, and the frequency of positive samples grown on group 2 cells (65.0%) was higher than the frequency of positive samples grown on group 1 cells (50.0%). The number of samples positive for adenoviruses was higher with A549 cells (13 samples) than with BGMK cells (one sample); the numbers of samples positive for enteroviruses were similar with both types of cells. By using phylogenetic analysis, adenoviral amplicons were grouped into subgenera A, C, D, and F, and enteroviral amplicons were grouped into coxsackieviruses B3 and B4 and echoviruses 6, 7, and 30, indicating that A549 and BGMK cells were suitable for recovering a wide range of adenoviral and enteroviral types. The cell culture-PCR assay with a combination of A549 and BGMK cells and molecular identification could be a useful tool for monitoring infectious adenoviruses and enteroviruses in aquatic environments.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.70.11.6695-6705.2004
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
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