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Firefly Luciferase Mutant with Enhanced Activity and Thermostability.

  • Author(s): Pozzo, Tania
  • Akter, Farhima
  • Nomura, Yoko
  • Louie, Angelique Y
  • Yokobayashi, Yohei
  • et al.
Abstract

The luciferase isolated from the firefly Photinus pyralis (Ppy) catalyzes a two-step reaction that results in the oxidation of d-luciferin accompanied by emission of yellow-green light with a peak at 560 nm. Among many applications, Ppy luciferase has been used extensively as a reporter gene in living cells and organisms. However, some biological applications are limited by the low stability of the luciferase and limited intracellular luciferin concentration. To address these challenges, efforts to protein engineer Ppy luciferase have resulted in a number of mutants with improved properties such as thermostability, pH tolerance, and catalytic turn over. In this work, we combined amino acid mutations that were shown to enhance the enzyme's thermostability (Mutant E) with those reported to enhance catalytic activity (LGR). The resulting mutant (YY5) contained eight amino acid changes from the wild-type luciferase and exhibited both improved thermostability and brighter luminescence at low luciferin concentrations. Therefore, YY5 may be useful for reporter gene applications.

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