A multifunctional single-attachment-point reagent for controlled protein biotinylation.
The biotin/avidin system is one of the most widely used affinity detection and affinity capture systems in biology. However, the determination of the exact number of biotin tags attached onto a substrate is complicated by the fact that biotin does not present any light-absorbing or -emitting properties. Here, we describe a fluorescent biotinylation reagent designed from the general multifunctional single-attachment-point (MSAP) reagent concept. A Lys-Cys dipeptide scaffold was used to display a biotin functional group and a fluorescein functional group along with an N-hydroxysuccinimide ester reactive group. The resulting bifunctional MSAP reagent, Fl-Biotin-NHS, was used to prepare a monobiotinylated version of cetuximab, which was further reacted with avidin to obtain a soluble avidin-based cetuximab oligomer. The MSAP peptide-scaffold approach allows fluorophores, chromophores, or reactive groups to be combined with biotin and provides a broad approach to obtain multifunctional biotin-based reagents.
Garanger
Elisabeth
E
Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Boston, Massachusetts 02129, USA.
Weissleder
Ralph
R
Josephson
Lee
L
D000577:Amides; D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000737:chemistry; D001360:Avidin; D019921:Biotinylation / Q000379:methods; D000068818:Cetuximab; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D005456:Fluorescent Dyes / Q000737:chemistry; D013386:Succinates
D000577:Amides; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D013386:Succinates; D001360:Avidin; D000068818:Cetuximab; C011116:succinamide