| Abstract
| - Recently we demonstrated that the fusion of an octapeptide to the C-terminus of a cysteine-free mutant ofaequorin showed no inhibitory effect on the luminescenceactivity of the photoprotein. This observation is of particular importance when the use of aequorin as a label inthe development of immunoassays for peptides whoseactivity lies in their C-terminal region or the epitope forantibody recognition is at their C-terminus is desired. Inthe case of opioid peptides, antibodies are directed towardtheir C-terminus as they differ from each other at thisterminus. The goal of this study was to develop animmunoassay for Leu-enkephalin, a mammalian opioidpeptide, using a C-terminal aequorin−peptide fusionprotein. For that, the N-terminus of Leu-enkephalin wasgenetically fused to the C-terminus of a cysteine-freemutant of aequorin. It was observed that the C-terminalconjugated aequorin maintained its luminescence activity.An immunoassay for Leu-enkephalin was then developedusing the aequorin−Leu-enkephalin fusion protein as alabeled analyte in a competitive as well as in a sequentialbinding mode. It was demonstrated that aequorin can beused as a label in peptide assays in which it is criticalthat the peptide's C-terminus be free for activity and/orfor antibody recognition.
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