title: Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity.
abstract: MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry's fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Yi
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Yingche
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Jiahui
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Photobiology and Bioimaging, The Scintillon Institute, San Diego, California, United States of America.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Shaner
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
mesh_terms: D000818:Animals; D037421:Anthozoa; D004926:Escherichia coli / Q000378:metabolism*; D019143:Evolution, Molecular; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D015723:Gene Library; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009841:Oligonucleotides; D000097573:Red Fluorescent Protein