title: Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.
abstract: Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.
authors.affiliation: †Department of Biosciences, Rice University, Houston, Texas 77005, United States.
authors.forename: Naresh
authors.initials: N
authors.lastname: Pandey
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Nobles
authors.forename: Lynn
authors.initials: L
authors.lastname: Zechiedrich
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Maresso
authors.affiliation: †Department of Biosciences, Rice University, Houston, Texas 77005, United States.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Silberg
mesh_terms: D005453:Fluorescence; D050939:Gene Fusion / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics*; D010455:Peptides / Q000235:genetics; D060066:Protein Interaction Maps / Q000235:genetics*; D019265:Spectroscopy, Near-Infrared / Q000379:methods
chemical_list: D008164:Luminescent Proteins; D010455:Peptides
keywords: gene fission; gene fusion; mutagenesis; near-infrared fluorescent protein; protein fragment complementation; protein−protein interaction