Electroextraction of Insoluble Proteins from the Organic Matrix of the Nacreous Layer of the Japanese Pearl Oyster, Pinctada fucata

• Published in: Methods and protocols 2019-05, Vol.2 (2), p.37
• Main Authors: Funabara, Daisuke, Miyashita, Natsumi, Nagai, Kiyohito, Maeyama, Kaoru, Kanoh, Satoshi
• Format: Article
• Language: English
• Subjects: Electro-extracted proteins; electroextraction; heating method; nacreous layer; organic matrix; pearl oyster
• ISSN: 2409-9279; 2409-9279
• DOI: 10.3390/mps2020037

The nacreous layer of shells and pearls is composed of aragonite crystals arranged in an organic matrix. The organic matrix contains chitin and several proteins that regulate the formation of the nacreous layer. Owing to their strong interactions in the organic matrix, the current method for extraction of insoluble proteins from the pre-powdered nacreous layer involves heating to high temperatures in the presence of a detergent (e.g., sodium dodecyl sulfate, SDS) and reductant (e.g., dithiothreitol, DTT), which is likely to induce protein degradation. Therefore, we have developed an electroextraction method to isolate proteins from the organic matrix of a nacreous organic sheet, that was obtained following the decalcification of shells in their original shape. Our electroextraction method employs milder conditions without heating or detergent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the electro-extracted proteins (EEPs) under non-reduced and reduced conditions revealed that this method yielded a greater number of different proteins compared with the conventional extraction method and the isolated EEPs retained their disulfide bonds. Our method is able to easily extract insoluble proteins from the nacreous layer under mild conditions and will undoubtedly aid future analyses into the functions of the nacreous layer proteins.