An improved method of protein isolation and proteome analysis with <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Laminariales) incubated under different pH conditions |
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Authors: | Eun-Young Kim Dong-Gyun Kim Yu-Ri Kim Hye-Jung Hwang Taek-Jeong Nam In-Soo Kong |
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Institution: | (1) Department of Biotechnology, Pukyong National University, Busan, South Korea;(2) Department of Food Science and Biotechnology, Pukyong National University, Busan, South Korea; |
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Abstract: | The brown alga Saccharina japonica is abundant on rocky coasts of Far East Asia, including Korea, Japan, and China. S. japonica produces high levels of compounds used in the food, cosmetic, and pharmaceutical industries. Thus, many studies have focused
on the biosynthesis, extraction, purification, and application of carbohydrates, as well as biochemical features that yield
cellular proteins. However, total protein isolation has proved difficult, due to viscous polysaccharides on the surface of
S. japonica. To extract total proteins cleanly from S. japonica, we examined various lysis buffers and detergents for effective cell lysis and removal of polysaccharide. Lysis solution
D (7 M urea, 4% 3-(3-cholami-dopropyl dimethylammonio) propanesulfonate], 2 M thio-urea, 100 mM dithiothreitol, 4% pharmalyte,
4% polyvinylpyrrolidone) achieved a comparatively high yield of protein extraction, with 12 mg of proteins purified per 1 g
of dry weight of S. japonica. Proteins isolated using lysis solution D and subjected to two-dimension polyacrylamide gel electrophoresis generated more
than 200 protein spots. Of these, 60 spots were analyzed by matrix-assisted laser desorption ionization-time of flight/mass
spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS. A database search revealed that these proteins include glyceraldehyde-3-phosphate
dehydrogenase, tryptophan synthase α chain, 6-phosphogluconate dehydrogenase (6PGD), actin, phosphoglycerate kinase, elongation
factor Tu, kinesin, fucoxanthin-chlorophyll a–c binding protein F precursor and ATP synthase subunit β. Many protein spots
were unidentified. When S. japonica was incubated at different pH, tryptophan synthase α chain and variant surface glycoprotein 7 precursor were highly expressed
at pH 7.5 and 9.5, respectively, whereas 6PGD and kinesin showed low expression at pH 9.5. |
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