Dual labeling of a binding protein allows for specific fluorescence detection of native protein
2001 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 295, no 1, 22-30 p.Article in journal (Refereed) Published
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 Asn(23)Cys 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-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 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 Fc(3(1)), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.
Place, publisher, year, edition, pages
2001. Vol. 295, no 1, 22-30 p.
biosensor, fluorescent labeling, fluorescence resonance energy transfer, protein engineering staphylococcal protein A, affibody, antibody variable domains, bacterial receptor domain, taq dna-polymerase, energy-transfer, combinatorial libraries, immunoassay, conformation, mutagenesis, fragments, affibody
IdentifiersURN: urn:nbn:se:kth:diva-20852ISI: 000170298900004OAI: oai:DiVA.org:kth-20852DiVA: diva2:339549
QC 201005252010-08-102010-08-10Bibliographically approved