Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein.
Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-naphthalenesulfonic acid (EDANS) and 6-(7-nitrobenzofurazan-4-ylamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.
Engfeldt
Torun
T
Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, 10691 Stockholm, Sweden.
Renberg
Björn
B
Brumer
Harry
H
Nygren
Per Ake
PA
Karlström
Amelie Eriksson
AE
D001665:Binding Sites; D015374:Biosensing Techniques; D001710:Biotin / Q000378:metabolism; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D007070:Immunoglobulin A / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis; D017433:Protein Structure, Secondary; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
D005456:Fluorescent Dyes; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D011506:Proteins; D001710:Biotin