Dual Labeling of a Binding Protein Allows for Specific Fluorescence Detection of Native Protein

• Published in: Analytical biochemistry Vol. 295; no. 1; pp. 22 - 30
• Main Authors: Karlström, Amelie, Nygren, Per-Åke
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2001
• Subjects: affibody; antibody variable domains; bacterial receptor domain; biosensor; combinatorial libraries; conformation; Energy Transfer; Fluorescence; fluorescence resonance energy transfer; Fluorescent Dyes; fluorescent labeling; fragments; immunoassay; Models, Molecular; mutagenesis; Mutagenesis, Site-Directed; Protein Binding; Protein Conformation; protein engineering; protein engineering staphylococcal protein A; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - chemistry; Spectrometry, Fluorescence - methods; staphylococcal protein A; Staphylococcal Protein A - analysis; Staphylococcal Protein A - chemistry; Staphylococcal Protein A - genetics; taq dna-polymerase; fluorescence resonance energy transfer; protein engineering; biosensor; affibody; fluorescent labeling; staphylococcal protein A
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1006/abio.2001.5186

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.