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 staphylococcal protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn23Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) 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 Fc region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of Fc3(1) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc3(1), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.
Karlström
A
A
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, S-100 44, Sweden.
Nygren
P A
PA
D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013205:Staphylococcal Protein A / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A