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

Index: Bioorg. Med. Chem. Lett. 20(23) , 6905-8, (2010)

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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 Ni(2+), 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.Copyright © 2010 Elsevier Ltd. All rights reserved.