ArticleJournal of visualized experiments : JoVE2018
Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence.
Article in Journal of visualized experiments : JoVE, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
4 citing papers in PubMed.
- Residue-Specific Incorporation of Noncanonical Amino Acids in Auxotrophic Hosts:Chemical reviews · 2025Review
- Advances in Biosynthesis of Non-Canonical Amino Acids (ncAAs) and the Methods of ncAAs Incorporation into Proteins.Molecules (Basel, Switzerland) · 2023Review
- Combating Antimicrobial Resistance With New-To-Nature Lanthipeptides Created by Genetic Code Expansion.Frontiers in microbiology · 2020Review
- Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications.Applied microbiology and biotechnology · 2019Review
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Authors and funding
7 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore's conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced "cyan" variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.
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