Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology(1,2). When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function 3. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive(4,5). The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein DsRed'' by directed evolution first to increase the speed of maturation(6), then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions(7). Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.