1. Solid-phase synthesis of imprinted nanoparticles as artificial antibodies against the C-terminus of the cannabinoid CB1 receptor: exploring a viable alternative for bioanalysis
- Author
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Nora Unceta, Alberto Gómez-Caballero, Antonio Guerreiro, Gontzal García del Caño, Ramón J. Barrio, Joan Sallés, Miquel Saumell-Esnaola, M. Aranzazu Goicolea, and Ainhoa Elejaga-Jimeno
- Subjects
chemistry.chemical_classification ,Original Paper ,CB1 receptor ,medicine.diagnostic_test ,Chemistry ,C-terminus ,molecularly imprinted nanoparticles ,bioanalysis ,Dot blot ,Fusion protein ,Epitope ,Analytical Chemistry ,Amino acid ,GPCR ,Biochemistry ,Western blot ,Receptor, Cannabinoid, CB1 ,artificial antibody ,epitope imprinting ,medicine ,Target protein ,G protein-coupled receptor - Abstract
[EN]The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to G(i/o) proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 +/- 10.5 nm at pH 3 (25 oC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 +/- 15.2 nm at 35 degrees C. Lower critical solution temperature of this polymer was found to be approximate to 33.4 degrees C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1(414-472) and GST-CB1(414-442) were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR). Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for this research was provided by the Spanish Ministry of Science, Innovation and Universities (project CTQ2017-85686-R) and by the Basque Government (Research Groups of the Basque University System, Project No IT 1186-19.
- Published
- 2021