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      FACS-optimized mutants of the green fluorescent protein (GFP).

      1 , ,

      Gene

      Elsevier BV

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          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.

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          Author and article information

          Journal
          Gene
          Gene
          Elsevier BV
          0378-1119
          0378-1119
          1996
          : 173
          : 1 Spec No
          Affiliations
          [1 ] Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305-5402, USA. cormack@cmgm.stanford.edu
          Article
          0378111995006850
          10.1016/0378-1119(95)00685-0
          8707053
          30a0f91e-b958-4c25-9ab4-73bcb7c1835c

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