| Abstrakt: |
A modified human β2receptor, designated 0K-β2, was developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes. 0K-β2has the following modifications: (1) all 16 lysines in the wild-type β2receptor 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-β2receptor, the amino terminal amine and the ϵ-amine of Lys3, both reside in the amino-terminal FLAG epitope. The 0K-β2receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type β2receptor. 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-β2receptor. Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the 0K-β2receptor, a lysine-depleted β2receptor that retains functional characteristics of the wild-type receptor. |
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