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. 2008 May 21;130(20):6488-97.
doi: 10.1021/ja7114579. Epub 2008 Apr 29.

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

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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 et al. J Am Chem Soc. .

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, beta-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.

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