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Proceedings Paper

Application to processing system using intra-molecular BRET
Author(s): Tomomi Otsuji; Emiko Okuda-Ashitaka; Satoshi Kojima; Hidehumi Akiyama; Seiji Ito; Yoshihiro Ohmiya
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Paper Abstract

Luciferases are used as the reporter gene for promoter activity, whereas a green fluorescent protein (GFP) is used as marker for cellular function and localization. Recently, bioluminescence resonance energy transfer (BRET) between luciferase and YFP is used for analysis of inter-molecular reaction such as ligand-receptor in the living cells. The neuropeptides nocistatin (NST) and nociceptin/orphanin FQ (Noc/OFQ) are derived from the same precursor protein, while NST exhibits antagonism against Noc/OFQ-actions. In this study, we attempt an intra-molecular BRET system for monitoring dynamic biological process of the production of NST and Noc/OFQ in the living cells. At first, we constructed a fusion protein (Rluc-GFP) covalently linking luciferase (Renilla luciferase; Rluc) to Aequorea GFP as an intra-molecular BRET partner. Furthermore, we inserted constructs of mouse NST and Noc/OFQ (Rluc-m-GFP) or bovine NST and Noc/OFQ (Rluc-b-GFP) containing a proteolytic cleavage motif (Lys-Arg) within Rluc-GFP. When these constructions were transfected into Cos7 cells, all fusion proteins had luciferase activity and specific fluorescence. Luminescence spectra of Rluc-GFP, Rluc-m-GFP and Rluc-b-GFP fusion proteins with DeepBlueC as a substrate showed two peaks centered at 400 nm and 510 nm, whereas Rluc showed one peak centered at 400 nm. These results indicate that the proteolytic cleavage motif inserted fusion proteins between luciferase and GFP are available for intra-molecular BRET systems at first step.

Paper Details

Date Published: 12 September 2003
PDF: 8 pages
Proc. SPIE 4967, Genetically Engineered and Optical Probes for Biomedical Applications, (12 September 2003); doi: 10.1117/12.477879
Show Author Affiliations
Tomomi Otsuji, National Institute of Advanced Industrial Science and Technology (Japan)
Emiko Okuda-Ashitaka, Kansai Medical Univ. (Japan)
PRESTO, Japan Science and Technology Corp. (Japan)
Satoshi Kojima, National Institute of Advanced Industrial Science and Technology (Japan)
Osaka Univ. (Japan)
Hidehumi Akiyama, Univ. of Tokyo (Japan)
Seiji Ito, Kansai Medical Univ. (Japan)
Yoshihiro Ohmiya, National Institute of Advanced Industrial Science and Technology (Japan)
PRESTO, Japan Science and Technology Corp. (Japan)


Published in SPIE Proceedings Vol. 4967:
Genetically Engineered and Optical Probes for Biomedical Applications
Darryl J. Bornhop; Ramesh Raghavachari; Samuel I. Achilefu; Alexander P. Savitsky, Editor(s)

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