RACE-based amplification of cDNA and expression of a luciferin-regenerating enzyme (LRE): An attempt towards persistent bioluminescent signal

Abstract

In the firefly light organ, luciferin-regenerating enzyme (LRE) plays an important role in the recycling of oxyluciferin into luciferin. In this study, the LRE cDNA was cloned from total RNA extracted from lantern of Lampyris turkestanicus (an Iranian firefly) using PCR-based techniques including reverse transcription-polymerase chain reaction, 5′-RACE (5′-rapid amplification of cDNA ends) and 3′-RACE. The full-length cDNA encoding LRE of L. turkestanicus (T-LRE) was 1047 with 924 bp open-reading frame encoding the protein of 307 amino acid residues with a calculated molecular mass of 33.5 kDa. The deduced amino acid sequence of T-LRE showed 46 and 45% identity to A-LRE (LRE from the American firefly – Photinus pyralis) and G-LRE (LRE from a Japanese firefly – Luciola cruciata), respectively. The cDNA encoding LRE of L. turkestanicus was expressed in recombinant Escherichia coli and it is shown that T-LRE improves the luminescent signal of firefly luciferase.