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

In this study, we generated blue- and red-shifted ( −16 and 12 nm) luminescence-emittingCypridina 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  × 106 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 andRenilla 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.

Source: Photochemistry and Photobiology - September 17, 2023 Category: Science Authors: Yoshiyuki Nishimiya, Yosuke Morita, Chun Wu, Yasushi Ohyama, Yuki Tochigi, Tsugumi Okuzawa, Mami Sakashita, Atsushi Asakawa, Tsutomu Irie, Yoshihiro Ohmiya, Satoru Ohgiya, Naoki Morita Tags: RESEARCH ARTICLE Source Type: research