Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments

Abstract

Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O 2 , and added luciferin. Previous Photinus pyralis red-emitting variants with high K m values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a K m value similar to CBR and ~2.6-fold higher specific activity.The red-emitting PLR3 was ~2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the K m value in low ATP environments. KeywordsBioluminescence; firefly; luciferase; ATP; red-emitting; imaging Bioluminescent proteins, epitomized by the beetle luciferases (Lucs), are now proven reagents for noninvasive imaging studies. As reporters, bioluminescent enzymes can visualize genetic activity and many other cellular biochemical events; while in living animals, they can be used to track specific types of cells including tumors [1][2][3][4][5]. Major reasons for the current success of bioluminescence imaging (BLI) include: the great detectability (signal to noise) due to essentially nonexistent endogenous background; wide dynamic range; and the availability of reasonably priced commercial CCD-based detection devices [1][2][3]. Moreover, a particular advantage of the beetle Lucs, which produce light by oxidizing the luciferin substrate (LH 2 ) in reactions that also require Mg-ATP and molecular * To whom correspondence should be addressed: Tel.: + 1 860 439-2479, brbra@conncoll.edu. 1 Abbreviations used: BLI, bioluminescence imaging; CBR, Promega's click beetle red, recombinant Pyrophorus plagiophthalamus luciferase (GenBank: AY258591); LH 2 , D-firefly luciferin; Luc, luciferase; Luc2, Promega's Photinus pyralis-based luciferase (GenBank: AY738222); PLG2, recombinant P. pyralis luciferase variant (GenBank: KY486507); PLR3, recombinant P. pyralis luciferase variant (GenBank: KY486508); PpyRE9, recombinant P. pyralis luciferase variant (GenBank: GQ404466); and RLU, relative light units. All luciferases were expressed from the human codon optimized sequences indicated above. Competing interests statementThe authors declare no competing interests. Mainly for reasons of improved detectability that can be achieved by the more efficient transmission of light through animal tissues, light emission at wavelengths greater than 600 nm is highly advantageous [7,8] for good expression and stability at 37 °C, the specific activity was ~3.5-fold lower and the emission maxima was slightly blue-shifted (Table 1). Importantly, we succeeded in reducing both K m values: ~30-fold for Mg-ATP (3-fold greater than that for CBR) and ~7-fold for LH 2 (~2-fold lower than the CBR value). The mutations primarily responsible for the redshifted emission were Y255F and S284T, while the lowered K m values resulted from the G246A, F250H, and V351I amino acid changes that also contributed to the relative drop in...

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