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Titolo:
Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing
Autore:
Ozawa, T; Kaihara, A; Sato, M; Tachihara, K; Umezawa, Y;
Indirizzi:
Japan Sci & Technol Corp, JST, Tokyo, Japan Japan Sci & Technol Corp Tokyo Japan & Technol Corp, JST, Tokyo, Japan Univ Tokyo, Sch Sci, Dept Chem, Bunkyo Ku, Tokyo 1130033, Japan Univ Tokyo Tokyo Japan 1130033 ept Chem, Bunkyo Ku, Tokyo 1130033, Japan
Titolo Testata:
ANALYTICAL CHEMISTRY
fascicolo: 11, volume: 73, anno: 2001,
pagine: 2516 - 2521
SICI:
0003-2700(20010601)73:11<2516:SLAAOP>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
INTERACTIONS IN-VIVO; RECRUITMENT SYSTEM; FIREFLY LUCIFERASE; GENETIC SYSTEM; DNAE GENE; PHOSPHORYLATION; COMPLEMENTATION; EXPRESSION; UBIQUITIN; INDICATOR;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Physical, Chemical & Earth Sciences
Citazioni:
29
Recensione:
Indirizzi per estratti:
Indirizzo: Umezawa, Y Japan Sci & Technol Corp, JST, Tokyo, Japan Japan Sci & TechnolCorp Tokyo Japan Corp, JST, Tokyo, Japan
Citazione:
T. Ozawa et al., "Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing", ANALYT CHEM, 73(11), 2001, pp. 2516-2521

Abstract

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly Luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp, PCC6803 wasused: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaEintein, wherein the protein splicing occurs and thereby the extein of Ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin sen-ed to monitor the interaction bem een IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity, This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.

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Documento generato il 21/09/20 alle ore 17:25:28