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.
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.
authors.affiliation: Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Porter
authors.forename: Cliff I
authors.initials: CI
authors.lastname: Stains
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Jester
authors.forename: Indraneel
authors.initials: I
authors.lastname: Ghosh
mesh_terms: D002474:Cell-Free System; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D049409:Luciferases, Firefly / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D012333:RNA, Messenger; C518603:protein kinase inhibitor peptide (5-24); D012313:RNA; D004247:DNA; D049409:Luciferases, Firefly; D017868:Cyclic AMP-Dependent Protein Kinases; D022021:Tacrolimus Binding Proteins