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Nat Methods. 2006 Dec;3(12):985-93. Epub 2006 Oct 29.

Monitoring regulated protein-protein interactions using split TEV.

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1
Max Planck Institute of Experimental Medicine, Hermann Rein Str. 3, D-37075 G├Âttingen, Germany.

Abstract

Signaling cascades integrate extracellular stimuli primarily through regulated protein-protein interactions (PPIs). Intracellular signal transduction strictly depends on PPIs occurring at the membrane and in the cytosol. To monitor constitutive and regulated protein interactions within living mammalian cells, we have developed a biological assay termed split TEV. We engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins. Functional reconstitution of TEV protease fragments can be monitored with 'proteolysis-only' reporters, which can be previously silent fluorescent and luminescent reporter proteins. Additionally, proteolytically cleavable inactive transcription factors can be combined with any downstream reporter gene of choice to yield 'transcription-coupled' reporter systems. Thus, split TEV combines the advantages of split enzyme- and reporter gene-mediated assays, and provides full flexibility with regard to the final readout. In a first biological application, we monitored neuregulin-induced ErbB2/ErbB4 receptor tyrosine kinase heterodimerization.

PMID:
17072307
DOI:
10.1038/nmeth967
[Indexed for MEDLINE]
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