Construction of a 'turn-on' fluorescent probe system for His-tagged proteins

Atsushi Murata, Satoshi Arai, Su In Yoon, Masao Takabayashi, Miwako Ozaki, Shinji Takeoka

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Hexahistidine ((His)6) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)6, and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni2+, can bind (His)6. The system is turned off when Dabcyl-(His)6 is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)6-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)6-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His) 6-protein.

Original languageEnglish
Pages (from-to)6905-6908
Number of pages4
JournalBioorganic and Medicinal Chemistry Letters
Volume20
Issue number23
DOIs
Publication statusPublished - 2010 Dec 1

Keywords

  • Energy transfer
  • Fluorescent probe
  • Histidine-tag
  • Molecular recognition

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry

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