Abstract
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°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 μg 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 −1CDW (K1E RNAP) of the reporter protein Gfp was produced in shaking flask cultures of B. megaterium.
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Acknowledgement
This work was financially supported by “Deutsche Forschungsgemeinschaft (SFB578)”.
The genomic phage DNA of K1E and SP6 bacteriophage was kindly provided by Dr. Martina Mühlenhoff, Dr. Rita Gerardy-Schahn, Dr. Graham Hatfull, and Dr. Roger Hendrix, respectively. Thanks to Dr. Mark G. Carter for the plasmids of the pNIAysic series and to Dr. Richard Münch for the support during the computational part of this work. Furthermore, thanks to Katrin B. Müller and Jannika Viereck for the scientific assistance during this work. Thanks to Stefanie Klein for the preparation of T7 RNA polymerase.
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Stammen, S., Schuller, F., Dietrich, S. et al. Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium . Appl Microbiol Biotechnol 88, 529–539 (2010). https://doi.org/10.1007/s00253-010-2732-y
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DOI: https://doi.org/10.1007/s00253-010-2732-y