Glow‐type conversion and characterization of a minimal luciferase via mutational analyses

Author:

Ohmuro‐Matsuyama Yuki1ORCID,Matsui Hayato1,Kanai Masaki1,Furuta Tadaomi2ORCID

Affiliation:

1. Technology Research Laboratory Shimadzu Corporation Kyoto Japan

2. School of Life Science and Technology Tokyo Institute of Technology Yokohama Japan

Abstract

Luciferases are widely used as reporter proteins in various fields. Recently, we developed a minimal bright luciferase, picALuc, via partial deletion of the artificial luciferase (ALuc) derived from copepods luciferases. However, the structures of copepod luciferases in the substrate‐bound state remain unknown. Moreover, as suggested by structural modeling, picALuc has a larger active site cavity, unlike that in other copepod luciferases. Here, to explore the bioluminescence mechanism of picALuc and its luminescence properties, we conducted multiple mutational analyses, and identified residues and regions important for catalysis and bioluminescence. Mutations of residues likely involved in catalysis (S33, H34, and D55) markedly reduced bioluminescence, whereas that of residue (E50) (near the substrate in the structural model) enhanced luminescence intensity. Furthermore, deletion mutants (Δ70–Δ78) in the loop region (around I73) exhibited longer luminescence lifetimes (~ 30 min) and were reactivated multiple times upon re‐addition of the substrate. Due to the high thermostability of picALuc, one of its representative mutant (Δ74), was able to be reused, that is, luminescence recycling, for day‐scale time at room temperature. These findings provide important insights into picALuc bioluminescence mechanism and copepod luciferases and may help with sustained observations in a variety of applications.

Publisher

Wiley

Subject

Cell Biology,Molecular Biology,Biochemistry

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