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    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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    Author
    Pandey, Naresh; Nobles, Christopher L.; Zechiedrich, Lynn; Maresso, Anthony W.; Silberg, Jonathan J.
    Date
    2015
    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.
    Citation
    Pandey, 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." ACS Synthetic Biology, 4, no. 5 (2015) American Chemical Society: 615-624. http://dx.doi.org/10.1021/sb5002938.
    Published Version
    http://dx.doi.org/10.1021/sb5002938
    Keyword
    gene fission; gene fusion; mutagenesis; near-infrared fluorescent protein; protein fragment complementation; More... protein?protein interaction Less...
    Type
    Journal article
    Publisher
    American Chemical Society
    Citable link to this page
    https://hdl.handle.net/1911/88284
    Rights
    This 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.
    Link to License
    http://pubs.acs.org/page/policy/authorchoice_termsofuse.html
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    • BioSciences Publications [365]
    • Faculty Publications [4988]

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    Home | FAQ | Contact Us | Privacy Notice | Accessibility Statement
    Managed by the Digital Scholarship Services at Fondren Library, Rice University
    Physical Address: 6100 Main Street, Houston, Texas 77005
    Mailing Address: MS-44, P.O.BOX 1892, Houston, Texas 77251-1892
    Site Map