Molecular evolution of Cypridina noctiluca secretory luciferase for production of spectrum‐shifted luminescence‐emitting mutants and their application in nuclear receptor–reporter assays

Abstract Luciferase is a popular enzyme used for biological analyses, such as reporter assays. In addition to a conventional reporter assay using a pair of firefly and Renilla luciferases, a simple multicolor reporter assay using multiple firefly or beetle luciferases emitting different color luminescence with a single substrate has been reported. Secretory luciferases have also been used for convenient sample preparation in reporter assays; however, reporter assay using secretory luciferase mutants that emit spectrum‐shifted luminescence have not yet been reported. In this study, we generated blue‐ and red‐shifted (−16 and 12 nm) luminescence‐emitting Cypridina secretory luciferase (CLuc) mutants using multiple cycles of random and site‐directed mutagenesis. Even for red‐shifted CLuc mutant, which exhibited relatively low activity and stability, its enzymatic activity was sufficiently high for a luciferase assay (3.26 × 10 6 relative light unit/s), light emission was sufficiently prolonged (half‐life is 3 min), and stability at 37°C was high. We independently determined the luminescence of these CLuc mutants using a luminometer with an optical filter. Finally, we replaced the commonly used reporters, firefly and Renilla luciferases used in a conventional nuclear receptor–reporter assay with these CLuc mutants and established a secretory luciferase‐based single‐substrate dual‐color nuclear receptor–reporter assay.