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 language | English |
|---|---|
| Pages (from-to) | 6905-6908 |
| Number of pages | 4 |
| Journal | Bioorganic and Medicinal Chemistry Letters |
| Volume | 20 |
| Issue number | 23 |
| DOIs | |
| Publication status | Published - 2010 Dec 1 |
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Keywords
- Energy transfer
- Fluorescent probe
- Histidine-tag
- Molecular recognition
ASJC Scopus subject areas
- Pharmaceutical Science
- Drug Discovery
- Organic Chemistry
- Molecular Medicine
- Molecular Biology
- Clinical Biochemistry
- Biochemistry
Cite this
Construction of a 'turn-on' fluorescent probe system for His-tagged proteins. / Murata, Atsushi; Arai, Satoshi; Yoon, Su In; Takabayashi, Masao; Ozaki, Miwako; Takeoka, Shinji.
In: Bioorganic and Medicinal Chemistry Letters, Vol. 20, No. 23, 01.12.2010, p. 6905-6908.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Construction of a 'turn-on' fluorescent probe system for His-tagged proteins
AU - Murata, Atsushi
AU - Arai, Satoshi
AU - Yoon, Su In
AU - Takabayashi, Masao
AU - Ozaki, Miwako
AU - Takeoka, Shinji
PY - 2010/12/1
Y1 - 2010/12/1
N2 - 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.
AB - 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.
KW - Energy transfer
KW - Fluorescent probe
KW - Histidine-tag
KW - Molecular recognition
UR - http://www.scopus.com/inward/record.url?scp=78149286156&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78149286156&partnerID=8YFLogxK
U2 - 10.1016/j.bmcl.2010.10.011
DO - 10.1016/j.bmcl.2010.10.011
M3 - Article
C2 - 21035333
AN - SCOPUS:78149286156
VL - 20
SP - 6905
EP - 6908
JO - Bioorganic and Medicinal Chemistry Letters
JF - Bioorganic and Medicinal Chemistry Letters
SN - 0960-894X
IS - 23
ER -