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.

@article{citeulike:2951470, 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.}, address = {Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.}, author = {Porter, J. R. and Stains, C. I. and Jester, B. W. and Ghosh, I. }, citeulike-article-id = {2951470}, doi = {http://dx.doi.org/10.1021/ja7114579}, issn = {1520-5126}, journal = {Journal of the American Chemical Society}, keywords = {emsa, interactome, split-tev}, month = {May}, number = {20}, pages = {6488--6497}, posted-at = {2008-07-02 09:03:39}, priority = {2}, title = {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.}, url = {http://dx.doi.org/10.1021/ja7114579}, volume = {130}, year = {2008} }

TY - JOUR ID - citeulike:2951470 L3 - citeulike-article-id:2951470 TI - 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. JF - Journal of the American Chemical Society VL - 130 IS - 20 SP - 6488 EP - 6497 AD - Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA. SN - 1520-5126 N2 - 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. KW - emsa KW - interactome KW - split-tev AU - Porter, JR AU - Stains, CI AU - Jester, BW AU - Ghosh, I PY - 2008/05/21/ UR - http://dx.doi.org/10.1021/ja7114579 ER -