Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin : activities at the rat bradykinin B2 receptor

Authors: Charest-Morin, XavierLodge, RobertMarceau, François
Abstract: To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2 or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue™ (microscopy), a luminol-based reagent or TMB (luminescence or colorimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range 50-600 nM. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity and possible steric hindrance between the receptor and large proteins.
Document Type: Article de recherche
Issue Date: 7 February 2018
Open Access Date: 15 February 2018
Document version: AM
This document was published in: Canadian Journal of Physiology and Pharmacology, (2018)
N R C Research Press
Alternative version:
Collection:Articles publiés dans des revues avec comité de lecture

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