title: Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3.
abstract: Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Yasunaga
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Murotomi
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Abe
authors.affiliation: Tsuruga Institute of Biotechnology, TOYOBO Corporation Ltd., Tsuruga, Fukui 914-0047, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Tsuruga Institute of Biotechnology, TOYOBO Corporation Ltd., Tsuruga, Fukui 914-0047, Japan.
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Nishii
authors.affiliation: Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Yonago, Tottori 683-8503, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ohbayashi
authors.affiliation: Chromosome Engineering Research Center, Tottori University, Yonago, Tottori 683-8503, Japan; Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Sciences, Tottori University, Yonago, Tottori 683-8503, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.affiliation: Department of Psychiatry and Center for Circadian Biology, University of California, San Diego, La Jolla, CA 92093-0603, USA.
authors.forename: Takako
authors.initials: T
authors.lastname: Noguchi
authors.affiliation: National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8063, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Niwa
authors.affiliation: DAILAB, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8063, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohmiya
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan. Electronic address: y-nakajima@aist.go.jp.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nakajima
mesh_terms: D000818:Animals; D002154:Calpain / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D009124:Muscle Proteins; D008156:Luciferases; C105884:CAPN3 protein, human; D002154:Calpain
keywords: Calpain 3; Destabilization sequence; Luciferase; Real-time monitoring