title: Simultaneous assembly of two target proteins using split inteins for live cell imaging.
abstract: Inteins are protein elements that covalently reassemble proteins from two precursor fragments in a process known as protein splicing. They are commonly used to reassemble a single target protein by protein splicing, but a second target protein can potentially reassemble by intein dimerization. Here, we use the naturally occurring split DnaE intein from Nostoc punctiforme (NpuDnaE) to demonstrate the simultaneous assembly of two target proteins in several examples studied with live cell imaging: yellow fluorescent protein (YFP) with monomeric red fluorescent protein (mRFP), dominant positive mutant of RhoA GTPase with YFP and GCaMP2 Ca(2+) indicator with mRFP. These examples showed the versatility of the strategy along with some interesting attributes: first, the two target proteins are in equal stoichiometry; second, the extent of protein splicing can be reported by a fluorescent protein. In particular, the split GCaMP2 with mRFP could find applications in tissue-specific Ca(2+) imaging in transgenic organisms, where mRFP could control for motion-related intensity changes.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Stanley
authors.initials: S
authors.lastname: Wong
authors.forename: Evan
authors.initials: E
authors.lastname: Mills
authors.forename: Kevin
authors.initials: K
authors.lastname: Truong
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D019281:Dimerization; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D047668:Inteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D046937:Nostoc / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D020742:rhoA GTP-Binding Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D002118:Calcium; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D020742:rhoA GTP-Binding Protein; D000097573:Red Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria