As a class, fluorescent proteins have been subjected to more extensive protein engineering and artificial directed evolution than almost any other category of protein. Such a concentrated effort on these probes is due to the fact that fluorescent proteins are extremely popular tools in the biological sciences and improved variants can provide huge benefits to researchers. Although an impressive degree of progress in fluorescent protein development has been made to date, the temptation to say that the current fluorescent protein palette is good enough should be actively resisted. In most cases, current fluorescent protein variants are good enough to meet the demands of many current applications; in all probability these proteins will not perform as needed in future applications.
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology 22: 1567-1572 (2004). The first report of what has now been termed the "mFruit" series of fluorescent proteins derived from mRFP1. Nathan Shaner and associates describe mCherry, mOrange, tdTomato, mStrawberry and several additional variants.
Shaner, N. C., Lin, M. Z., McKeown, M. R., Steinbach, P. A., Hazelwood, K. L., Davidson, M. W., and Tsien R. Y.
Improving the photostability of bright monomeric orange and red fluorescent proteins. Nature Methods 5: 545-551 (2008). Additional mutagenesis efforts on mOrange and TagRFP to produce variants having improved photostability. The authors introduce a new screen targeting photostability and succeed in producing several of the most photostable fluorescent proteins yet reported.
Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24: 79-88 (2006). Dr. Geoffrey Waldo and associates report a new variant of GFP that contains a number of folding mutations designed to prevent mis-folding of the fluorescent protein when fused to insoluble partners. The authors also introduce these mutations into blue, cyan, and yellow variants.
Dai, M., Fisher, H. E., Temirov, J., Kiss, C., Phipps, M. E., Pavlik, P., Werner, J. H., and Bradbury, A. R.
The creation of a novel fluorescent protein by guided consensus engineering. Protein Engineering, Design and Selection 20: 69-79 (2007). The authors describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. The result, named CGP (consensus green protein), was well expressed, monomeric, and brighter than Azami Green, the closest relative.
Molecular replacement by evolutionary search. Acta Crystallography D 57: 1474-1479 (2001). A discussion of stochastic search algorithms that are used to perform rapid six-dimensional molecular-replacement techniques to optimize the development of new proteins. The authors discuss the performance of the algorithm and its dependence on search model quality and target function.
Evolutionary optimization of fluorescent proteins for intracellular FRET. Nature Biotechnology 23: 355-360 (2005). A clever application of evolutionary design principles to apply fluorescence activated cell sorting in order to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting variants, named YPet and CyPet, exhibited substantially higher dynamic range than the precursor proteins.
Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochemical Journal 400: 531-540 (2006). The authors engineered a monomeric version of a tetrameric cyan fluorescent protein from Clavularia using a designed synthetic gene library. The new variant, termed mTFP1, exhibits cyan-green emission, a high degree of photostability, and impressive brightness.
Directed evolution of an extremely stable fluorescent protein. Protein Engineering, Design and Selection 22: 313-323 (2009). Extension of the work on CGP (consensus green protein) to develop a variant that could not be denatured by a standard thermal melt and preserved almost full fluorescence after overnight incubation at 80 ° C. This approach may be generally applicable to the stabilization of other proteins.
Evolution of new nonantibody proteins via iterative somatic hypermutation. Proceedings of the National Academy of Sciences (USA) 101: 16745-16749 (2004). Development of a novel technique known as somatic hypermutation to create monomeric red fluorescent proteins with increased photostability and far-red emission, thus surpassing the best efforts of structure-based design.
Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library. Nature Biotechnology 24: 1569-1571 (2006). The authors describe using a library targeting residues neighboring the chromophore to develop an enhanced blue fluorescent protein (named Azurite) that exhibits enhanced quantum yield, reduced pH sensitivity, and a 40-fold increase in photobleaching half-life.