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Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging†

  1. Xueyan Li1,
  2. Yoshihiro Nakajima1,
  3. Kazuki Niwa1,
  4. Vadim R. Viviani2,
  5. Yoshihiro Ohmiya1,*

Article first published online: 28 OCT 2009

DOI: 10.1002/pro.279

Issue

Protein Science

Protein Science

Volume 19, Issue 1, pages 26–33, January 2010

Additional Information

How to Cite

Li, X., Nakajima, Y., Niwa, K., Viviani, V. R. and Ohmiya, Y. (2010), Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging. Protein Science, 19: 26–33. doi: 10.1002/pro.279

Author Information

  1. 1

    Cell Dynamics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka 563-8577, Japan

  2. 2

    Laboratório de Bioquímica e Biotecnologia de Sistemas Bioluminescentes, Universidade Federal de São Carlos, Campus de Sorocaba, Sorocaba, SP, Brazil

*1-8-31 Midorigaoda, Ikeda, Osaka 563-8577, Japan,

  1. †

    The authors declare no conflicts of interest.

Publication History

  1. Issue published online: 6 JAN 2010
  2. Article first published online: 28 OCT 2009
  3. Accepted manuscript online: 28 OCT 2009 12:00AM EST
  4. Manuscript Accepted: 9 OCT 2009
  5. Manuscript Received: 10 JUL 2009

Funded by

  • NEDO grant (Chemical Substance Management Technology Projects of Development of Simple and Highly Functional Hazard Assessment Methods)
  • The Ministry of Economy, Trade and Industry of Japan

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Keywords:

  • red-emitting luciferase;
  • enhanced activity;
  • thermostable mutant;
  • bioassays;
  • bioimaging

Abstract

A luciferase from the railroad worm (Phrixothrix hirtus) is the only red-emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site-directed mutagenesis, we produced red-emitting mutants with higher activity and better stability. Compared with the wild-type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8-fold in I212L/N351K, 8.4-fold in I212L, and 7.8-fold in I212L/S463R; and the cell-based activities were 3.6-fold in I212L/N351K and 3.4-fold in N351K. The remaining activities after incubation at 37°C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell-based BLI was performed, and the luminescence signal was 3.6-fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.

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