Isolation and expression of a cDNA encoding Renilla reniformis luciferase

Key findings

Lorenz et al. cloned a 1.2-kbp cDNA from a Renilla reniformis lambdagt11 library encoding a 314-amino acid, 34 kDa luciferase protein. The ORF was identified using two degenerate 17-base oligonucleotide probes derived from native V8-protease peptide sequences; clone ARLuc-6 hybridized to both probes and was subcloned into pTZ18R for expression.

Expression of recombinant luciferase from pTZRLuc-1 in IPTG-induced E. coli yielded 2 x 10^15 hv/sec/ml bioluminescence activity, 7-fold greater than uninduced cells. SDS/PAGE confirmed a 34 kDa band co-migrating with native luciferase, and immunoblotting with polyclonal anti-luciferase antibody confirmed protein identity.

Recombinant luciferase catalyzed coelenterazine oxidation with blue bioluminescence (lambda-max = 480 nm) and transferred energy to Renilla GFP, shifting emission to green (lambda-max = 509 nm), matching native enzyme characteristics. Activity in E. coli demonstrated that N-linked glycosylation is not required for catalytic function, validating recombinant Renilla luciferase as functionally equivalent to the native protein.