New monomeric
variants of DsRed.
Daniel E. Strongin,
Brooke J. Bevis, Benjamin S. Glick
Molec. Genetics and Cell Bio., University of Chicago, 920 E. 58th St., Chicago,
IL 60637, USA (destrong@uchicago.edu)
The red fluorescent protein DsRed promises to be an important and versatile tool for cell biology. Like its structural homologue GFP, DsRed can be genetically fused to a protein of interest. DsRed has an excitation maximum at 558 nm and an emission maximum at 583 nm. At these wavelengths, phototoxicity and cellular autofluorescence are low. DsRed can be excited by common laser lines and detected with standard filter sets. Advances by our lab and others have enhanced the utility of DsRed by producing variants with fast fluorophore maturation and a reduced tendency to form higher-order aggregates. However, a major limitation of DsRed is that it oligomerizes to form a stable tetramer, and as a result, many DsRed fusion proteins are nonfunctional or even toxic. Campbell et al (PNAS 99:7877-7882) recently reported the creation of a monomeric DsRed called mRFP1. Despite this advance, mRFP1 has limitations, including reduced brightness and rapid photobleaching. Moreover, mRFP1 is red-shifted relative to wild-type DsRed, and thus poorly suited to FRET with GFP. Using a combination of site directed and random mutagenesis, we have created new monomeric DsRed variants in an ongoing effort to optimize this fluorescent protein as a research tool.