| Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium. | |
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MedLine Citation:
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PMID: 20596705 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Gene "7" of Escherichia coli phage K1E was proposed to encode a novel DNA-dependent RNA polymerase (RNAP). The corresponding protein was produced recombinantly, purified to apparent homogeneity via affinity chromatography, and successfully employed for in vitro RNA synthesis. Optimal assay conditions (pH 8, 37 degrees C, 10 mM magnesium chloride and 1.3 mM spermidine) were established. The corresponding promoter regions were identified on the phage genome and summarized in a sequence logo. Surprisingly, next to K1E promoters, the SP6 promoter was also recognized efficiently in vitro by K1E RNAP, while the T7 RNAP promoter was not recognized at all. Based on these results, a system for high-yield in vitro RNA synthesis using K1E RNAP was established. The template plasmid is a pUC18 derivative, which enables blue/white screening for positive cloning of the target DNA. Production of more than 5 microg of purified RNA per microgram plasmid DNA was achieved. Finally, in vivo protein production systems for Bacillus megaterium were established based on K1E and SP6 phage RNAP transcription. Up to 61.4 mg g (CDW) (-1) (K1E RNAP) of the reporter protein Gfp was produced in shaking flask cultures of B. megaterium. |
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Authors:
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Simon Stammen; Franziska Schuller; Sylvia Dietrich; Martin Gamer; Rebekka Biedendieck; Dieter Jahn |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2010-07-02 |
Journal Detail:
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Title: Applied microbiology and biotechnology Volume: 88 ISSN: 1432-0614 ISO Abbreviation: Appl. Microbiol. Biotechnol. Publication Date: 2010 Sep |
Date Detail:
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Created Date: 2010-08-23 Completed Date: 2011-01-25 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8406612 Medline TA: Appl Microbiol Biotechnol Country: Germany |
Other Details:
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Languages: eng Pagination: 529-39 Citation Subset: IM |
Affiliation:
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Institute of Microbiology, Technische Universität Braunschweig, Germany. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Bacillus megaterium
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metabolism* Coliphages / enzymology* DNA-Directed RNA Polymerases / genetics, isolation & purification, metabolism* RNA / biosynthesis*, chemistry Recombinant Proteins / genetics, metabolism* Templates, Genetic Viral Proteins / genetics, metabolism* |
| Chemical | |
Reg. No./Substance:
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0/Recombinant Proteins; 0/Viral Proteins; 63231-63-0/RNA; EC 2.7.7.6/DNA-Directed RNA Polymerases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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