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In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase

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Abstract

We have previously shown that, in vivo, the integration system based on the gene encoding the TG1 integrase and the corresponding attB TG1 and attP TG1 sites works well not only in Streptomyces strains, but also in Escherichia coli. Furthermore, the attachment sites for TG1 integrase are distinct from those of ϕC31 integrase. In this report, we expressed TG1 integrase as a GST-TG1 integrase fusion protein and then used affinity separation and specific cleavage to release purified integrase. Conditions for in vitro recombination were established using the purified TG1 integrase and its cognate attP TG1 and attB TG1 sites. TG1 integrase efficiently catalyzed a site-specific recombination between attB TG1 and attP TG1 sites irrespective of their substrate topology. The minimal sequences of attP TG1 and attB TG1 sites required for the substrates of TG1 integrase were demonstrated to be 43 and 39-bp, respectively. These results provide the basic features of the TG1 integrase system to be used as biotechnological tools, as well as to unravel the mechanism of the serine integrase.

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Acknowledgments

We thank H. Shindo and A. Yamashita for their technical support. We also acknowledge Prof. T. Beppu and M. Shirai for their interest in the study. This work was supported by Grants-in-Aid for Scientific Research, the 21st Century COE program and the High-Tech Research Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Author notes

  1. Nobutaka Hirano

    Present address: Department of Materials Chemistry and Engineering, College of Engineering, Nihon University, Koriyama, Fukushima, 963-8642, Japan

Authors and Affiliations

  1. Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa, 252-8510, Japan

    Kentaro Morita, Tomoyuki Yamamoto, Naoki Fusada, Nobutaka Hirano & Hideo Takahashi

  2. The Kitasato Institute for Life Science, Kitasato University, Kanagawa, 28-8555, Japan

    Mamoru Komatsu & Haruo Ikeda

Authors
  1. Kentaro Morita
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  2. Tomoyuki Yamamoto
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  3. Naoki Fusada
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  4. Mamoru Komatsu
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  5. Haruo Ikeda
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  6. Nobutaka Hirano
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  7. Hideo Takahashi
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Corresponding author

Correspondence to Hideo Takahashi.

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Communicated by A. Aguilera.

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Morita, K., Yamamoto, T., Fusada, N. et al. In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase. Mol Genet Genomics 282, 607–616 (2009). https://doi.org/10.1007/s00438-009-0490-2

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  • DOI: https://doi.org/10.1007/s00438-009-0490-2

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