A Tandem Green-Red Heterodimeric Fluorescent Protein with High FRET Efficiency

Chembiochem. 2016 Dec 14;17(24):2361-2367. doi: 10.1002/cbic.201600492. Epub 2016 Nov 15.

Abstract

The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato-a tandem fusion of a dimeric DsRed variant. Here we describe the engineering of brighter red (RRvT), green (GGvT), and green-red heterodimeric (GRvT) tdTomato variants. GRvT exhibited 99 % intramolecular FRET efficiency, resulting in long Stokes shift red fluorescence. These new variants could prove useful for multicolor live-cell imaging applications.

Keywords: FRET; dimerization; directed evolution; fluorescence; protein engineering.

MeSH terms

  • Animals
  • Anthozoa / metabolism
  • Dimerization
  • Fluorescence Resonance Energy Transfer*
  • HeLa Cells
  • Humans
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification

Substances

  • Luminescent Proteins
  • Recombinant Proteins
  • red fluorescent protein