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Reporter Genes02:11

Reporter Genes

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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
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Red fluorescent proteins engineered from green fluorescent proteins.

Hiromi Imamura1, Shiho Otsubo2, Mizuho Nishida1

  • 1Division of Systemic Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.

Proceedings of the National Academy of Sciences of the United States of America
|October 23, 2023
PubMed
Summary

Researchers engineered green fluorescent proteins (GFPs) into red fluorescent proteins (RFPs) by introducing specific mutations. This work clarifies the mechanism of red fluorophore formation and enables new fluorescent protein development.

Keywords:
X-ray crystallographyfluorescent proteinslive imagingprotein engineering

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Fluorescent proteins (FPs) are crucial biological tools, with green FPs (GFPs) and red FPs (RFPs) exhibiting distinct fluorophore structures.
  • Engineering GFPs into RFPs is challenging due to low sequence homology and unclear mechanisms governing their autocatalytic reactions.

Purpose of the Study:

  • To elucidate the mechanism of red fluorophore formation by converting GFPs to RFPs.
  • To identify key mutations and structural rearrangements required for green-to-red fluorescent protein conversion.

Main Methods:

  • Site-directed mutagenesis of coral GFPs (AzamiGreen and mcavGFP) to generate RFPs.
  • Structural comparison of the ancestral GFP and the derived RFP (AzamiRed1.0).
  • Assessment of a monomeric RFP variant for cellular labeling in mammalian cells.

Main Results:

  • Successful conversion of GFPs into RFPs through defined mutations.
  • Identified drastic rearrangements in amino acid interactions around the fluorophore, necessitating coordinated multisite mutations.
  • Demonstrated the creation of an oxygen-entry cavity essential for red fluorophore maturation.
  • Showcased the utility of a monomeric RFP for labeling cellular components.

Conclusions:

  • Coordinated multisite mutations are critical for converting GFPs to RFPs.
  • Structural rearrangements facilitate red fluorophore formation by enabling oxygen incorporation.
  • Protein engineering of GFPs offers a viable strategy for developing novel RFPs for biotechnological applications.