Mutational optimization of the coelenterazine-dependent luciferase from Renilla

Abstract

Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved kcat, as well as elevated luminescence in Arabidopsis. This advances the utility of RLUC as a reporter protein, biosensor, or resonance energy donor.

Figure 1

Half-life of RLUC enzyme activity. (A) Time course of in vivo luminescence in E. coli cells expressing wild type RLUC and two P220 mutants. (B) Time course of in vitro luminescence of wild type RLUC and two P220 mutants. Enzyme and substrate concentrations were 10 nM and 2 μM respectively in PBS buffer pH7.2. Absolute activities were normalized for better comparison (first time-point = 100). Results shown are averages from three biological replicates each performed in triplicate. The Coomassie stained SDS-PAGE gels below show that the P220 mutants and wild-type RLUC accumulate to equivalent levels in E. coli. RLU stands for relative luminescence units.

Figure 2

Decay properties of RLUC enzyme activity. (A) A titration assay indicates that 1 molecule of RLUC can turn over ~100 molecules of coelenterazine. A 1 μM solution of native coelenterazine substrate was incubated with increasing amounts of RLUC enzyme (1 nM to 100 nM) and the reaction was allowed to go to completion. Once light emission had ceased, the reaction was split in half and supplemented with fresh enzyme (squares) to examine whether all substrate had been depleted; or with fresh substrate (circles) to check for residual enzyme activity. Note, if an excess of substrate was added, the loss of RLUC activity was not reversible by adding fresh substrate. (B) Spectrophotometric confirmation of substrate turnover by RLUC. Absorbance spectra were recorded for 10 μM coelenterazine before (black trace) and after (blue trace) turnover by 100 nM RLUC. Two traces of partially completed reactions are shown for comparison (red and green traces). (C) Half life of RLUC activity in the in vitro assay as measured by luminescence decay kinetics.

Figure 3

Enzyme kinetics of optimized RLUC proteins. (A) Derivation of K_M values. The K_M for wild-type RLUC was similar to previously published data such as 210 nM (coelenterazine h)⁵ and 300 nM²³. Also shown are wild-type RLUC, RLUC+ and SuperRLUC, which were purified by nickel affinity chromatography and run on a polyacrylamide gel. (B) Luminescence spectra for RLUC, RLUC+ and Super-RLUC. (C) Time course of luminescence activity. Note the increased half life of activity of the RLUC+ and SuperRLUC mutants. (D) Inhibition of RLUC activity by high substrate concentration.

Figure 4

Improvement of light emission by SuperhRLUC in Arabidopsis and recombination vectors. (A) In vivo luminescence measurement of transgenic Arabidopsis expressing regular RLUC (2 lines), hRLUC, or SuperhRLUC. Asterisk represents the significant increase of luciferase activity of SuperhRLUC over hRLUC (P < 0.01, two tailed t-test; n = 4 repeats with 25 seedlings each). The immunoblot below probed with RLUC antibody confirms that hRLUC and SuperhRLUC (36 kDa) accumulate to similar, high, levels; the original RLUC can only be detected on the immunoblot after prolonged development. The arrowhead indicates a non-specific immunoreaction. (B) Photon-counting images of representative seedling roots. Photon emission in the primary roots of SuperhRLUC transgenic Arabidopsis was stronger compared to regular hRLUC (lower panels). The upper panels show a 3-dimensional version of the images below in which photon intensity is encoded in the third axis. Seedlings were incubated in 2 μM coelenterazine and imaged for 5 min. (C) pBS-SuperhRLUC-attR and pBS-attR-SuperhRLUC are recombination vectors for expression of SuperhRLUC fusion proteins, which contain the lambda att recombination sites utilized by the Gateway™ (Invitrogen) system [25]. Sequence elements flanking an insert (target cDNA) are shown before (pENTR) and after (Destination) attL × attR recombination. The 35S indicates a strong promoter in plants, which can be replaced by restriction digestion with KpnI and SwaI or AvrII. The ccdB gene provides for counter-selection of non-recombinants.