Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed).
The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with green fluorescent protein (GFP). But wild-type DsRed has several drawbacks, including slow chromophore maturation and poor solubility. To overcome the slow maturation, we used random and directed mutagenesis to create DsRed variants that mature 10-15 times faster than the wild-type protein. An asparagine-to-glutamine substitution at position 42 greatly accelerates the maturation of DsRed, but also increases the level of green emission. Additional amino acid substitutions suppress this green emission while further accelerating the maturation. To enhance the solubility of DsRed, we reduced the net charge near the N terminus of the protein. The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast.
Bevis
Brooke J
BJ
The University of Chicago, Department of Molecular Genetics and Cell Biology, 920 East 58th Street, Chicago, IL 60637, USA.
Glick
Benjamin S
BS
D000818:Animals; D001216:Asparagine / Q000737:chemistry; D003063:Cnidaria; D005821:Genetic Techniques; D005973:Glutamine / Q000737:chemistry; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D008164:Luminescent Proteins; C413663:fluorescent protein 583; D005973:Glutamine; D001216:Asparagine