Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions

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dc.citation.firstpage615en_US
dc.citation.issueNumber5en_US
dc.citation.journalTitleACS Synthetic Biologyen_US
dc.citation.lastpage624en_US
dc.citation.volumeNumber4en_US
dc.contributor.authorPandey, Nareshen_US
dc.contributor.authorNobles, Christopher L.en_US
dc.contributor.authorZechiedrich, Lynnen_US
dc.contributor.authorMaresso, Anthony W.en_US
dc.contributor.authorSilberg, Jonathan J.en_US
dc.date.accessioned2016-01-29T21:44:39Zen_US
dc.date.available2016-01-29T21:44:39Zen_US
dc.date.issued2015en_US
dc.description.abstractGene 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ﾖprotein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and proteinﾖprotein interactions within whole animals.en_US
dc.identifier.citationPandey, Naresh, Nobles, Christopher L., Zechiedrich, Lynn, et al.. "Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions." <i>ACS Synthetic Biology,</i> 4, no. 5 (2015) American Chemical Society: 615-624. http://dx.doi.org/10.1021/sb5002938.en_US
dc.identifier.doihttp://dx.doi.org/10.1021/sb5002938en_US
dc.identifier.urihttps://hdl.handle.net/1911/88284en_US
dc.language.isoengen_US
dc.publisherAmerican Chemical Societyen_US
dc.rightsThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.en_US
dc.rights.urihttp://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlen_US
dc.subject.keywordgene fissionen_US
dc.subject.keywordgene fusionen_US
dc.subject.keywordmutagenesisen_US
dc.subject.keywordnear-infrared fluorescent proteinen_US
dc.subject.keywordprotein fragment complementationen_US
dc.subject.keywordprotein?protein interactionen_US
dc.titleCombining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactionsen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
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