FACS-OPTIMIZED MUTANTS OF THE GREEN FLUORESCENT PROTEIN (GFP)

We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly reg ulated inducible promoter. We introduced random amino acid (aa) substi tutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FAGS) to select variants of GFP that fluoresce between 20- and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analys is reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima, In addition , when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the muta nts useful for a number of applications.