Design, expression, and characterization of a synthetic human cannabinoid receptor and cannabinoid receptor/ G-protein fusion protein†

摘要: We report here the synthesis and characterization of two gene constructs designed to facilitate structure/function studies human neuronal cannabinoid receptor, CB1. The first gene, which we call shCB1, is a synthetic containing unique restriction sites spaced roughly 50-100 bases apart rapid mutagenesis cloning. A nine amino acid epitope tag (from rhodopsin C-terminus) also present in shCB1 C-terminal tail enable detection purification using monoclonal antibody 1D4. find that can be transiently expressed COS cells with yield approximately 10-15 micro g receptor per 15 cm plate wild type like its ability bind ligands. Our confocal microscopy indicate targets membrane HEK293 internalized response agonist. To functional studies, made chimera C-terminus was fused N-terminus G-protein alpha subunit, Galphai. shCB1/Galphai shows agonist stimulated GTPgammaS binding, thus provides simplified way measure induced CB1 activation. Taken together, provide useful tools for biochemical biophysical examinations structure, activation attenuation.