A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters

Jason R. Porter, Cliff I. Stains, Benjamin W. Jester, Indraneel Ghosh

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, β-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12-48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein-protein, protein-DNA, and protein-RNA interactions in homogeneous assays with very high sensitivity (22-1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein-nucleic acid interactions and in evaluating antagonists of protein-protein and protein-peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein-protein and protein-nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.

Original languageEnglish (US)
Pages (from-to)6488-6497
Number of pages10
JournalJournal of the American Chemical Society
Volume130
Issue number20
DOIs
StatePublished - May 21 2008

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RNA
DNA
Proteins
Cell-Free System
Nucleic acids
Luciferases
Nucleic Acids
Purification
Artificial Cells
Green Fluorescent Proteins
Emulsions
Cell culture
Transfection
Peptides
Assays
Oils

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters. / Porter, Jason R.; Stains, Cliff I.; Jester, Benjamin W.; Ghosh, Indraneel.

In: Journal of the American Chemical Society, Vol. 130, No. 20, 21.05.2008, p. 6488-6497.

Research output: Contribution to journalArticle

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