Abstract
Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition. We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.
Highlights
Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level
We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride
From a random mutant library, this study identified two novel Photinus pyralis mutant luciferase enzymes that were found to retain their activities under dialysis condition
Summary
Luciferase, which generates bioluminescence during the oxidative decarboxylation of D-luciferin in the presence of ATP, has been used for numerous applications such as measuring biomass and cellular conditions (Lundin 2000) and protein-protein interaction (Christopoulos and Chiu 1995; OhmuroMatsuyama and Ueda 2016), assaying ATP-related enzymes (Clarke 2005; Lundin 2000), examining Dluciferin-generating enzymes using peptide-modified luciferin or luciferin derivatives (Cali et al 2006; Liu et al 2005; Noda et al 2010; O'Brien et al 2008), and performing real-time ATP imaging (Grygorczyk et al 2019). The bioluminescence-based methods have numerous applications, their sensitivity is reduced by the presence of various salts. The considerable loss in the sensitivity of the bioluminescence-based method limits the applications in the presence of salts
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