The calcium-binding photoprotein clytin II: Expression of the preferred human codon-optimized clytin II gene in Chinese hamster ovary-K1 cells and its use in the G-protein-coupled receptor assays

Abstract

Clytin II (CLII) is a Ca²⁺-binding photoprotein and has been identified as an isotype of clytin I (CLI). CLII consists of apoCLII (an apoprotein) and 2-peroxide of coelenterazine (an adduct of molecular oxygen to coelenterazine), which is identical to the widely used Ca²⁺-binding photoprotein, aequorin (AQ). However, CLII triggered by Ca²⁺ exhibits a 4.5-fold higher maximum luminescence intensity (I_max) compared to both AQ and CLI, and it is approximately 5 times less sensitive to Ca²⁺ than AQ. To confirm the suitability of the preferred human codon-optimized CLII (pCLII) gene for cell-based G-protein-coupled receptor (GPCR) assays, a transformant stably expressing apoprotein of pCLII using the pCLII gene in the mitochondria of CHO-K1 cells was established and in situ regenerated pCLII in the cells were applied to the high-throughput screening system. An ATP-stimulated GPCR assay for endogenous P2Y purinergic receptors was confirmed using the established stable transformant.

Keywords: Aequorin; Coelenterazine; Human cytochrome c oxidase; Mitochondria; P2Y purinergic receptors.