High through-put mutagenesis and screening methods were developed during Phase 1 for isolating spectral and kinetic variants of the Green Fluorescent Protein (GFP). Mutants were found which show large spectral shifts and accelerated fluorophore formation relative to wild-type. Optical screening of approximately 500,000 combinatorial mutants near the chromophore site yielded two main classes of proteins with blue- shifted emission (BFP) or red-shifted excitation (RSGFP). By shifting the excitation maximum from 390 to 490 nm, the RSGFP spectrum is well separated from cellular autofluorescence, making it vastly superior to wild-type GFP for epifluorescence microscopy and fluorescence activated cell sorting (FACS). In Phase 2, we plan to search for red-shifted emission derivatives of RSGFP using a method capable of negotiating very rough fitness landscapes. RSGFP and a red-shifted emission derivative would constitute an extremely valuable pair of molecular reagents, making two channel FACS, dual color epifluorescence microscopy, and fluorescence resonance energy transfer measurements readily available to cell biologists. In conjunction with imaging spectroscopy, the construction of a series of spectroscopically distinct red-shifted emission derivatives (e.g., green, yellow, orange, and red) would enable simultaneous analysis of several tagged promoters, fusion proteins, and/or cell types.
The isolation of a red-emitting derivative of the green Fluorescent Protein will constitute a highly visible flagship product for our company. From the standpoint of Phase 3 commercialization, this will result in: 1) direct sales of these molecular reagents (about $8,000,000 gross), 2) revenues from licensing composition of matter patent rights, 3) enhanced sales of imaging spectrophotometers, 4) an increase in in- house imaging contracts, and 5) enhanced sales of KAIROS' protein engineering software.
