Expression, purification, and characterization of recombinant apoPholasin

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Pholasin is a photoprotein composed of apoPholasin and an unknown chromophore.
• ApoPholasin's role in bioluminescence and its interaction with chromophores remain incompletely understood.

Purpose of the Study:

• To investigate the biochemical activity of apoPholasin.
• To characterize the luminescence produced by apoPholasin and coelenterazine.
• To identify the oxidation product of coelenterazine catalyzed by apoPholasin.

Main Methods:

• Human codon-optimized apoPholasin gene expression in mammalian cells.
• Detection of secreted apoPholasin using an anti-recombinant antibody.
• Expression and purification of glutathione S-transferase-apoPholasin (GST-apoPholasin) in bacteria via cold induction.
• Assay of luminescence activity with coelenterazine (CTZ).
• Spectroscopic analysis of bioluminescence emission and product fluorescence.
• Identification of CTZ oxidation products using analytical techniques.

Main Results:

• Secreted apoPholasin catalyzed CTZ oxidation, yielding continuous luminescence.
• Purified GST-apoPholasin exhibited luminescence activity with CTZ, with an emission peak at 461 nm.
• The luminescence product displayed purple-blue fluorescence under UV light (365 nm).
• Coelenteramine (CTM) was unexpectedly identified as the primary oxidation product of CTZ, rather than coelenteramide (CTMD).

Conclusions:

• ApoPholasin possesses intrinsic luminescence-catalyzing activity.
• The study elucidates a novel bioluminescence pathway involving apoPholasin and CTZ.
• The identification of CTM as the main product provides new insights into the mechanism of pholasin-based bioluminescence.