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

Publication ,  Journal Article
Cormack, BP; Valdivia, RH; Falkow, S
Published in: Gene
1996

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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Published In

Gene

DOI

ISSN

0378-1119

Publication Date

1996

Volume

173

Issue

1 Spec No

Start / End Page

33 / 38

Location

Netherlands

Related Subject Headings

  • Structure-Activity Relationship
  • Mutagenesis
  • Molecular Sequence Data
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Gene Library
  • Flow Cytometry
  • Escherichia coli
  • Developmental Biology
  • DNA
 

Citation

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Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1 Spec No), 33–38. https://doi.org/10.1016/0378-1119(95)00685-0
Cormack, B. P., R. H. Valdivia, and S. Falkow. “FACS-optimized mutants of the green fluorescent protein (GFP).Gene 173, no. 1 Spec No (1996): 33–38. https://doi.org/10.1016/0378-1119(95)00685-0.
Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33–8.
Cormack, B. P., et al. “FACS-optimized mutants of the green fluorescent protein (GFP).Gene, vol. 173, no. 1 Spec No, 1996, pp. 33–38. Pubmed, doi:10.1016/0378-1119(95)00685-0.
Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33–38.
Journal cover image

Published In

Gene

DOI

ISSN

0378-1119

Publication Date

1996

Volume

173

Issue

1 Spec No

Start / End Page

33 / 38

Location

Netherlands

Related Subject Headings

  • Structure-Activity Relationship
  • Mutagenesis
  • Molecular Sequence Data
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Gene Library
  • Flow Cytometry
  • Escherichia coli
  • Developmental Biology
  • DNA