The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with green fluorescent protein (GFP)1. But wild-type DsRed has several drawbacks, including slow chromophore maturation and poor solubility2,3. 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.
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Thanks to Sergey Lukyanov for sharing unpublished data, to Hiromi Sesaki and Rob Jensen for providing the pCox4-DsRed expression plasmid, to Susan Lindquist for use of the spectrofluorometer, to Dan Strongin for assistance with Supplementary Figure 1A, and to members of the Glick lab for feedback on the manuscript. This work was supported by grants from the National Science Foundation (MCB-9875939) and the American Cancer Society (RPG-00-245-01-CSM).
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Bevis, B., Glick, B. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat Biotechnol 20, 83–87 (2002). https://doi.org/10.1038/nbt0102-83
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