Highly chromophoric dual-terminus labeling of an intrinsically disordered native eukaryotic protein of interest at nanoscale

Jung Min Kim, Baik Lin Seong, Jinho Jung

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

Chemical conjugation of purified proteins of interest (POIs) in Escherichia coli cells is effective for high expression but has limitations for highly chromogenic dual labeling of intrinsically disordered native proteins (IDNPs). Our probes can tag IDNPs using chemical conjugation during protein synthesis and folding while preserving biologically active structures in mammalian cells. We fluorescently labeled IDNPs in mammalian cells using pure fluorescent methionine and ATTO 565-biotin at the N-or C-terminus, respectively. The dual-labeled Tat protein was used as a model for IDNPs in HeLa cells and detected using Ni-NTA beads to estimate its highly chromogenic concentration. We also demonstrated highly chromogenic double labeling of genetically encoded fluorescent-Tat expression in eukaryotic cells using a single fluorescent dye pair with Förster resonance energy transfer (FRET) ratio and two-color correlation analysis. This study aims to solve native POI processing and achieve ultra-sensitive protein folding for biological and ecological applications at the nanoscale.

Original languageEnglish
Article number125396
JournalInternational Journal of Biological Macromolecules
Volume245
DOIs
Publication statusPublished - 2023 Aug 1

Bibliographical note

Publisher Copyright:
© 2023

Keywords

  • Bead assay
  • Dual-fluorescent probe
  • Eukaryotic protein labeling
  • Intrinsically disordered native protein (IDNP). Macromolecular crowding
  • Two-color correlation

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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