title: Split-Cre recombinase effectively monitors protein-protein interactions in living bacteria.
abstract: The ability of Cre recombinase to excise genetic material has been used extensively for genome engineering in prokaryotic and eukaryotic cells. Recently, split-Cre fragments have been described that advance control of recombinase activity in mammalian cells. However, whether these fragments can be utilized for monitoring protein-protein interactions has not been reported. In this work, we developed a protein-fragment complementation assay (PCA) based on split-Cre for monitoring and engineering pairwise protein interactions in living Escherichia coli cells. This required creation of a dual-fluorescent reporter plasmid that permits visualization of reconstituted Cre recombinase activity by switching from red to green in the presence of an interacting protein pair. The resulting split-Cre PCA faithfully links cell fluorescence with differences in binding affinity, thereby allowing the facile isolation of high-affinity binders based on phenotype. Given the resolution of its activity and sensitivity to interactions, our system may prove a viable option for poorly expressed or weakly interacting protein pairs that evade detection in other PCA formats. Based on these findings, we anticipate that our split-Cre PCA will become a highly complementary and useful new addition to the protein-protein interaction toolbox.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
authors.forename: Sean P
authors.initials: SP
authors.lastname: O'Brien
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
mesh_terms: D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D060066:Protein Interaction Maps / Q000235:genetics*; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D011506:Proteins; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases
keywords: Escherichia coli; Fluorescent proteins; Pairwise protein interactions; Protein engineering; Protein-complementation assay