Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity

PLoS One. 2017 Feb 27;12(2):e0171257. doi: 10.1371/journal.pone.0171257. eCollection 2017.

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.

MeSH terms

  • Animals
  • Anthozoa
  • Escherichia coli / metabolism*
  • Evolution, Molecular
  • Fluorescence
  • Fluorescent Dyes / chemistry
  • Gene Library
  • Luminescent Proteins / chemistry*
  • Models, Molecular*
  • Mutagenesis, Site-Directed
  • Oligonucleotides / genetics
  • Protein Engineering / methods*

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Oligonucleotides
  • red fluorescent protein

Grant support

Natural Sciences and Engineering Research Council of Canada (RGPIN 288338-2010) http://www.nserc-crsng.gc.ca, Alberta Innovates scholarship to YS (no grant number) http://www.albertainnovates.ca, and Canada Research Chairs (no grant number) http://www.chairs-chaires.gc.ca/home-accueil-eng.aspx.