SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation

Sci Rep. 2013;3:2629. doi: 10.1038/srep02629.

Abstract

Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Cell Line
  • Chlorocebus aethiops
  • Chromophore-Assisted Light Inactivation*
  • Gene Expression
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Molecular Weight
  • Reactive Oxygen Species / chemistry
  • Reactive Oxygen Species / metabolism

Substances

  • Luminescent Proteins
  • Reactive Oxygen Species
  • Green Fluorescent Proteins