Site-specific fluorescence labeling of the beta2 adrenergic receptor amino terminus.
Anal Biochem
; 254(1): 88-95, 1997 Dec 01.
Article
em En
| MEDLINE
| ID: mdl-9398350
ABSTRACT
A modified human beta2 receptor, designated 0K-beta2, was developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes. 0K-beta2 has the following modifications (1) all 16 lysines in the wild-type beta2 receptor were mutated to arginines, (2) a FLAG epitope preceded by a cleaved hemagglutinin signal sequence was fused to the amino terminus, and (3) a hexahistidine tail was added to the carboxyl terminus. The FLAG epitope and hexahistidine tail were added to facilitate purification while lysine to arginine mutations eliminate potential labeling sites for amine-reactive fluorescent probes. The remaining primary amines in the 0K-beta2 receptor, the amino terminal amine and the epsilon-amine of Lys3, both reside in the amino-terminal FLAG epitope. The 0K-beta2 receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type beta2 receptor. The modified receptor was labeled with fluorescamine, an amine-reactive fluorescent probe. Proteolysis with factor Xa showed that labeling was confined to the amino terminus of the 0K-beta2 receptor. Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the 0K-beta2 receptor, a lysine-depleted beta2 receptor that retains functional characteristics of the wild-type receptor.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Receptores Adrenérgicos beta 2
/
Corantes Fluorescentes
/
Histidina
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Estados Unidos