Isolation and expression of a cDNA encoding Renilla reniformis luciferase
Study of Renilla bioluminescence and energy transfer to GFP was limited since no luciferase gene had been cloned. This paper isolated a Renilla luciferase cDNA and expressed active recombinant luciferase in E. coli. It matched native blue emission and gave green light with GFP.
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.
Abstract
Renilla reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in Renilla results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5]. In vivo, the excited state luciferin-luciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence. In vitro, Renilla luciferase emits blue light in the absence of any green fluorescent protein. A Renilla cDNA library has been constructed in lambda gt11 and screened by plaque hybridization with two oligonucleotide probes. We report here the isolation and characterization of a luciferase cDNA and its gene product. The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics.