C6-Deoxy coelenterazine analogues as an efficient substrate for glow luminescence reaction of nanoKAZ: The mutated catalytic 19 kDa component of Oplophorus luciferase

• The codon-optimized gene for the mutated 19 kDa protein (nanoKAZ) is prepared.
• The expression of nanoKAZ is succeeded in E. coli cells and CHO-K1 cells.
• The recombinant nanoKAZ is highly purified from E. coli cells.
• The substrate specificity of nanoKAZ is examined using coelenterazine analogues.
• Newly synthesized 6h-f-coelenterazine is an efficient substrate for nanoKAZ.

The codon-optimized gene for the mutated 19 kDa protein (nanoKAZ), which is the catalytic component of Oplophorus luciferase, was expressed in Escherichia coli cells and the recombinant protein was highly purified. The secretory expression of nanoKAZ from CHO-K1 cells was performed by fusing the secretory signal peptide sequence of Gaussia luciferase to the amino-terminus of nanoKAZ. The substrate specificity for the purified nanoKAZ and the nanoKAZ secreted into the cultured medium was determined, indicating that bis-coelenterazine (bis-CTZ) and newly synthesized 6h-f-coelenterazine (6h-f-CTZ) are an efficient substrate for the glow luminescence reaction of nanoKAZ.