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

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, aequo...

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Bibliographic Details
Published in:Protein expression and purification 2024-08, Vol.220, p.106481-106481, Article 106481
Main Authors: Inouye, Satoshi, Sato, Jun-ichi, Sahara-Miura, Yuiko, Hisada, Sunao
Format: Article
Language:English
Subjects:
Aequorin
Animals
Calcium - metabolism
Calcium-Binding Proteins - genetics
Calcium-Binding Proteins - metabolism
CHO Cells
Codon - genetics
Coelenterazine
Cricetinae
Cricetulus
Gene Expression
Human cytochrome c oxidase
Humans
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Mitochondria
P2Y purinergic receptors
Receptors, G-Protein-Coupled - genetics
Receptors, G-Protein-Coupled - metabolism
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
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Summary: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 (Imax) 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. •The Ca2+-binding photoprotein clytin II with a high signal-to-noise ratio is applied to the assays.•Preferred human codon-optimized clytin II (pCLII) gene is expressed efficiently in CHO–K1 cells.•The pCLII gene with the signal peptide sequence is expressed in the mitochondria of CHO–K1 cells.•CHO–K1 cells stably expressing the pCLII gene in mitochondria are established for the GPCR assay.•ATP-stimulated GPCR assay of endogenous P2Y purinergic receptors are successfully performed.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2024.106481