Abstract
Biosensors provide a sophisticated and discriminating means of probing biomolecular interactions. Specific ligands such as peptides and proteins can be immobilized onto sensor surfaces by a number of means including covalent attachment via amine, thiol or aldehyde chemistry, capture via biotin-avidin interaction or the use of specific tags. We have devised a simple chemoselective ligation method to selectively conjugate an anchoring functionality onto N-terminal serine or threonine residues of peptides and proteins allowing them to be immobilised onto the sensor surface in a defined orientation. It is based on the specific reaction of the 1,2-aminothiol of cysteine with an aldehyde under acidic conditions to form a stable thiazolidine product. The carbonyl precursors are derived from the 1,2-aminoalcohols of Ser or Thr that can be selectively and rapidly converted to the aldehyde form by periodate oxidation. Biotinylation of the aldehyde is then achieved via simple conjugation with a novel water-soluble dipeptide that contains a lysine residue bearing an Nε-cysteine-derived 1,2-aminothiol and an Nα-biotin moiety. Use of this method allowed selective biotinylation of a native form of murine EGF (mEGF2-53) that has an N-terminal serine residue. This derivative was then immobilised onto a streptavidin biosensor surface, and the resultant surface activity compared with those obtained by immobilising recombinant human EGF or the soluble extracellular domain of the EGF receptor (sEGFR1-621) using amine coupling (NHS/EDC) chemistry.The surface recognised the recombinant sEGFR with a similar KD to that of human EGF immobilised using NHS/EDC chemistry, or if the receptor was immobilised and murine EGF injected.
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Wade, J.D., Domagala, T., Rothacker, J. et al. Use of thiazolidine-mediated ligation for site specific biotinylation of mouse EGF for biosensor immobilisation. Letters in Peptide Science 8, 211–220 (2001). https://doi.org/10.1023/A:1016256525951
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DOI: https://doi.org/10.1023/A:1016256525951