title: FACS-optimized mutants of the green fluorescent protein (GFP).
abstract: We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis 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 mutants useful for a number of applications.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305-5402, USA. cormack@cmgm.stanford.edu
authors.forename: B P
authors.initials: BP
authors.lastname: Cormack
authors.forename: R H
authors.initials: RH
authors.lastname: Valdivia
authors.forename: S
authors.initials: S
authors.lastname: Falkow
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D015723:Gene Library; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D013329:Structure-Activity Relationship
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA