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A dual-light reporter system to determine the efficiency of protein–protein interactions in mammalian cells

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journal contribution
posted on 23.03.2015, 12:42 by M. T. Nasim, R. C. Trembath
Methods for determining protein–protein interactions in mammalian cells typically rely on single reporter functions and are susceptible to variations between samples particularly in regard to levels of transcription, processing and translation. A method has been developed for determining protein–protein interactions in mammalian cells, which bypasses these variables confounding single reporter assays. The approach utilizes two units of gene expression linked to reporter functions that are interposed by a deactivation–activation unit in such a way that the downstream expression unit is switched off. Hence upstream expression occurs regardless of protein–protein interaction, leading to the production of the upstream reporter. In the event of protein–protein interactions, the downstream expression unit is switched on leading to dual reporter read outs. Thus, the ratio of the two reporter activities provides a measure to determine the efficiency of protein–protein interactions. To access the system we screened a mutant of BMPR2 where the interaction between BMPR-II and LIMK is abrogated. BMPR-II is a type II receptor of the TGFβ superfamily and plays a key role in the pathogenesis of familial pulmonary arterial hypertension. This system has potential for high-throughput screening of libraries (peptide, chemical, cDNA, etc.) to isolate agents that are capable of interfering with highly selective protein–protein interaction.

Funding

The project was supported by a programme grant from the British Heart Foundation (RG/2000012 to RCT). Funding to pay the Open Access publication charges for this article was provided by the University of Leicester.

History

Citation

Nucleic Acids Research 33 (7): e66

Version

VoR (Version of Record)

Published in

Nucleic Acids Research 33 (7): e66

Publisher

Oxford University Press (OUP)

issn

0305-1048

eissn

1362-4962

Copyright date

2005

Available date

23/03/2015

Publisher version

http://nar.oxfordjournals.org/content/33/7/e66

Language

en

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