Dual labeling of a binding protein allows for specific fluorescence detection of native protein

Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the s taphylococcal. protein A (SPA)-IgG interaction was designed, in which a sin gle domain was engineered to facilitate site-specific incorporation of fluo rophores. An Asn(23)Cys mutant of the B domain from SPA was expressed in Es cherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamin e (1,5-LAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl) amino) hexanoate (NBD-X, SE), respectively. Biosensor analysis of Purified doubly labeled protein showed that high-affinity binding to the Fe region o f IgG was retained. The fluorescence emission spectrum of the doubly labele d protein showed a shift in the relative emission of the two fluorophores i n the presence of FC3(1)) fragments, which bind specifically to the B domai n. In addition, the fluorescence emission ratio 480/525 nm was shown to inc rease with increasing concentration of Fc(3(1)), whereas the presence of a control protein did not affect the emission ratio over the same concentrati on range. (C) 2001 Academic Press.