Expressão, purificação e caracterização das luciferases de Cratomorphus distinctus (Lampyridae) e Euryopa clarindae (Phengodidae) e sondagem de suas aplicabilidades
Abstract
Luciferases are the enzymes that catalyze the bioluminescence reaction in organisms
such as beetles. Bioluminescence has been used for bioanalytical purposes for
decades, including ATP detection assays, enzymatic assays, microbiological
contamination of products in the food industry, as a reporter gene for pollutant
biosensors, intracellular pH and heavy metal indicators, and bioimaging. Our research
group has cloned the largest number and variety of beetle luciferases in the world,
some already having bioanalytical applications, however there are still several
luciferases and mutant forms with interesting properties that we have produced, but
which have not yet been well characterized and applied. With the aim of expanding the
range of applications, especially in biosensors for pH and potentially toxic metals, we
carried out the characterization of the pH-sensitive luciferases from the lampyrid C.
distinctus and the orange light-emitting luciferase from the phengodid E. clarindae. To
achieve this, we subcloned the luciferase cDNA from C. distinctus and E. clarindae into
a pCold vector, transformed E. coli BL21DE3 bacteria and expressed and purified the
two luciferases by affinity chromatography on nickel-agarose resin. The tests showed
that the C. distinctus luciferase has a high catalytic efficiency for ATP and lower for
LH2, when compared to other lampyrid luciferases, in addition to having excellent
sensitivity to pH and metals, especially mercury. The E. clarindae luciferase showed a
high KM for LH2 and low for ATP, when compared to luciferases from other phengodids.
Thus, C. distinctus luciferase has potential applicability as a mercury and pH sensor
protein; while the insensitive luciferase from E. clarindae has potential as a reporter
gene. Furthermore, the orange light-emitting luciferase from E. clarindae constitutes
another important model to understand the structure x bioluminescence spectra
relationship in coleopteran luciferases and the origin of the different bioluminescence
colors.
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