EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THREE BRAZILIAN BEETLE LUCIFERASES
A.J.Silva Neto1, T.L.Oehlmeyer1, F.T.Ogawa1, B. Venkatesh 3, Y. Ohmiya3, V. R. Viviani1,2
1 Dept. Biologia Celular e Molecular, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brazil; 2 Universidade Federal de São Carlos, Sorocaba, SP, Brazil; 3 Cell Dynamics Research Group, Osaka National Research Institute, Osaka, Japan
The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR; lmax= 546 nm) and red-emitting (PxRE; lmax= 623 nm) luciferases of railroad worms Phrixotrix viviani and P. hirtus, and the green emitting luciferase from the larval click beetle Pyrearinus termitilluminans (Pyt;lmax= 534 nm) which produce the most green shifted bioluminescence among beetle luciferases. These luciferases were expressed and purified, and their properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics, but the kinetics can be affected by different treatments. The substrate affinities and catalytic efficiency of PxRE luciferase (KMLH2= 2 mM; KMATP= 70 mM; kcat=2.10-2 s-1) were higher than those of PxGR luciferase (KMLH2= 13.5 mM; KMATP=330 mM; kcat= 0,012.10-2 s-1 ), indicating that the red emitting luciferase is more efficient than the green emitting luciferase. (Financial support: FAPESP and JSPS).
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