Expression, purification, and characterization of recombinant apoPholasin
Introduction
The luminescence system in the boring mollusk Pholas dactylus was first reported by Dubois in 1887 [1,2] and consists of two glycoproteins of pholasin (a reactive oxygen-sensitive photoprotein, 34.6 kDa) [3,4] and a peroxidase (a reactive oxygen-producing enzyme containing a copper ion with a dimeric form of 310 kDa) [2]. In the early 1970s, pholasin and the peroxidase were designated as a luciferin (substrate) and a luciferase (enzyme), respectively [2]. The luminescence reaction of pholasin was triggered by reactive oxygen species such as superoxide anion and hydroxyl radical [2]. At the present time, pholasin is classified as a photoprotein composing of apoPholasin (an apoprotein of pholasin) and a prosthetic group for the light emission source [2]. However, the structure of the prosthetic group in native pholasin has not been identified to date.
In 1990, Müller and Campbell described that the prosthetic group in pholasin is unrelated to coelenterazine (CTZ) (Fig. 1A) and Cypridina luciferin. Moreover, based on the fluorescence spectrum of extracts from P. dactylus, they proposed that a flavin-like moiety might be involved in the luminescence reaction [4]. In 2000, Dunstan et al. successfully cloned the cDNA of apoPholasin from P. dactylus and revealed that apoPholasin consists of 225 amino acid (aa) residues including a functional signal peptide sequence with 20 aa residues for secretion [4]. The secreted apoPholasin might be an acidic protein (205 aa, calculated pI = 4.04) with seven cysteine residues. When the cDNA of apoPholasin was expressed in Escherichia coli cells, apoPholasin was expressed as inclusion bodies [4]. In a eukaryotic expression system using insect cells, apoPholasin was successfully secreted into the cultured medium. Further, the regeneration to active pholasin from secreted apoPholasin was examined by incubation with the methanol extracts of P. dactylus [4]. In 2008, Kuse et al. proposed that the prosthetic group of pholasin is a covalent adduct of dehydrocoelenterazine (dCTZ) (Fig. 1A) through the thiol group of a cysteine residue in apoPholasin [5,6]. However, there is no direct evidence that the chromophore covalently binds to a cysteine residue in native pholasin.
To better understand the luminescence system of pholasin, the preferred human-codon optimized gene [7] of apoPholasin was chemically synthesized, and apoPholasin was expressed into the culture medium from Chinese hamster ovary-K1 (CHO–K1) cells. Fortunately, we found that apoPholasin secreted from CHO–K1 cells showed continuous weak luminescence with CTZ like a luciferase. In addition, a soluble form of apoPholasin was obtained as a fusion protein from E. coli cells using the cold induction system [8,9], and the luciferase-like activity of apoPholasin with CTZ was confirmed using the purified fusion protein. In this manuscript, the luminescence and fluorescence properties of recombinant apoPholasin with CTZ are characterized.
Section snippets
Materials
Coelenterazine (CTZ), h-coelenterazine (h-CTZ), bis-coelenterazine (bis-CTZ) and coelenteramine (CTM) were obtained from JNC Corp. (Tokyo, Japan), and 6h-Coelenterazine (6h-CTZ) was prepared as previously described [10] (Fig. 1B). Coelenteramide (CTMD) was kindly provided by Dr. O. Shimomura on May 29, 2002. Dehydrocoelenterazine (dCTZ) was synthesized by the condensation of p-hydroxyphenylpyruvic acid and coelenteramine (CTM), as previously reported [11]. Lyophilized native pholasin (0.2 mg,
apoPholasin expressed in CHO–K1 cells shows a luciferase-like activity with CTZ
To confirm the secretory expression of apoPholasin in mammalian cells, the pcDNA3-opPho expression vector possessing the preferred human codon-optimized gene for apoPholasin was prepared, transfected, and expressed transiently in CHO–K1 cells. As Ham's F-12 medium containing fetal bovine serum showed the luminescence activity with CTZ, serum-free MCDB 201 medium was chosen for cell culture. Interestingly, we found that the addition of CTZ to the culture medium stimulated luminescence intensity
Conclusion
For the first time, we found that recombinant apoPholasin shows a luciferase-like activity with CTZ. The soluble protein expression of apoPholasin in E. coli cells was performed as a GST fusion protein. Purified GST-apoPholasin catalyzes the oxidation of CTZ, resulting in an emission peak at 461 nm, and the main oxidation product of CTZ is CTM, but not CTMD.
CRediT authorship contribution statement
Satoshi Inouye: Formal analysis, Writing - original draft. Yuiko Sahara-Miura: Formal analysis. Mitsuhiro Nakamura: Formal analysis. Takamitsu Hosoya: Formal analysis, Writing - review & editing.
Declaration of competing interest
The authors have declared no financial interests.
Acknowledgments
The authors thank T. Watanabe and Dr. T. Nakama (Yokohama Research Center, JNC Co.) for the synthesis of dehydrocoelenterazine and thank Dr. T. Hirano (University of Electro-Communications) for valuable discussions of the mechanism to produce coelenteramine from coelenterazine.
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