title: A genetically encoded bioluminescent indicator for illuminating proinflammatory cytokines.
abstract: We introduce a method to evaluate the activities of cytokines based on the nuclear transport of NF-κB. A pair of bioluminescent indicators was made for conferring cytokine sensitivity to cervical carcinoma-derived HeLa cells. The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (RLuc) by protein splicing with a DnaE intein from Synechocystis sp. PCC6803. The bioluminescence intensity of thus reconstituted RLuc in the HeLa cells was used as a measure of the activities for cytokines. With the present method, we evaluated the activities of various cytokines based on the nuclear transport of NF-κB in human cervical carcinoma-derived HeLa cells carrying the indicators. The present approach to evaluating the activities of cytokines may provide a potential clinical value in monitoring drug activity and directing treatment for various diseases related with NF-κB. The method highlights the experimental procedure from our original publications, Anal. Biochem. 2006, 359, 147-149 and Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542. The summary of the method is: •Cytokine activities are determined within 2 h after stimulation.•Temporarily inactivated split-luciferase fragments are reconstituted by protein splicing.•Nucleartrafficking of NF-κB was illuminated for gauging the ligand-driven activity.
authors.affiliation: Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan.
authors.forename: Sung Bae
authors.initials: SB
authors.lastname: Kim
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan.
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Umezawa
keywords: Bioluminescence; Cytokine; Genetically encoded bioluminescent indicator based on protein splicing; Luciferase; Nuclear trafficking; Protein splicing assay (PSA); Tumor necrosis factor-α (TNF-α); nuclear factor-κB (NF-κB)