RRC ID 73748
Author Takemoto K, Matsuda T, Sakai N, Fu D, Noda M, Uchiyama S, Kotera I, Arai Y, Horiuchi M, Fukui K, Ayabe T, Inagaki F, Suzuki H, Nagai T.
Title SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation.
Journal Sci Rep
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.
Volume 3
Pages 2629
Published 2013-1-1
DOI 10.1038/srep02629
PII srep02629
PMID 24043132
PMC PMC3775092
MeSH 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
IF 3.998
DNA material SuperNova/pRSETB (RDB19866)