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

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.