Evolution of a fluorinated green fluorescent protein

Hyeon Yoo Tae, A. James Link, David A. Tirrell

Research output: Contribution to journalArticle

72 Scopus citations

Abstract

The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved ≈650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents.

Original languageEnglish (US)
Pages (from-to)13887-13890
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number35
DOIs
StatePublished - Aug 28 2007

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Directed evolution
  • Noncanonical amino acids
  • Protein engineering

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