Super RLuc8: A novel engineered Renilla luciferase with a red-shifted spectrum and stable light emission

Abstract

Renilla luciferase is a bioluminescent enzyme which is broadly used as a reporter protein in molecular biosensors. In this study, a novel luciferase with desired light emission wavelength and thermostability is reported. The results indicated that the new luciferase, namely super RLuc8, had a red-shifted spectrum and showed stable light emission. Super RLuc8 showed a 10-fold (p-value=0.0084) increase in the thermostability at 37°C after 20min incubation, in comparison to the native enzyme. The optimum temperature of the mutant increased from 30 to 37°C. Molecular dynamics simulation analysis indicated that the increased thermostability was most probably caused by a better structural compactness and more local rigidity in the regions out of the emitter site.

Identification of New Degrons in Streptococcus mutans Reveals a Novel Strategy for Engineering Targeted, Controllable Proteolysis

“…To test this, we created N-terminal luciferase fusions using both of the hybrid bdNEDD8+HdiR degron tags and compared their degradation kinetics with that of a transcriptionally depleted, inducible luciferase construct. Luciferase was chosen due to its low intrinsic stability at 37°C (Loening et al, 2006 ; Branchini et al, 2007 ; Ebrahimi et al, 2012 ; Rahnama et al, 2017 ), which provides a best-case scenario to reduce protein abundance via transcriptional depletion. At T 0 , we added xylose to the degradable luciferase strains to trigger bdNEDP1 expression, while simultaneously removing xylose from the inducible luciferase culture (i.e., washout) to initiate its transcriptional depletion.…”

Palette of Luciferases: Natural Biotools for New Applications in Biomedicine

“…Синий цвет и низкий квантовый выход биолюминесценции ограничивают ее использование в экспериментах in vivo [44], поскольку животные ткани сильно поглощают видимый свет за пределами «окна прозрачности» (600-900 нм). Для преодоления раз-личных ограничений методами случайного или сайтнаправленного мутагенеза получен ряд репортеров на основе RLuc с улучшенными свойствами: повышенной устойчивостью к инактивации сывороткой крови, повышенной яркостью, а также белки со спектрами, смещенными в длинноволновую область [44,[47][48][49]. Чтобы расширить сферу применения RLuc, разработаны более яркие аналоги целентеразина с красным спектром люминесценции [49,50].…”

New imaging probes to track cell fate: reporter genes in stem cell research

“…Their reactions do not require ATP and oxygen, but are limited by substrate penetration and light absorption; Thus, unfortunately, they are rarely utilized for in vivo studies [41,42]. To improve the in vivo imaging performance of Renilla luciferases Loening et al and Rahnama et al have engineered variants with a red-shifted spectrum (peak emission of 556 and 540nm), of which a substantial portion is above 600nm [77,78]. …”