Production and Purification of Secretory Simian Cytidine Monophosphate-N-acetylneuraminic Acid Hydroxylase Using Baculovirus-Protein Expression System
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- Takahashi Tadanobu
- Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka
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- Kawagishi Sawako
- Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka
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- Funahashi Hiroki
- Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka
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- Hayashi Nonoka
- Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka
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- Suzuki Takashi
- Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka
書誌事項
- タイトル別名
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- Production and Purification of Secretory Simian Cytidine Monophosphate-<i>N</i>-acetylneuraminic Acid Hydroxylase Using Baculovirus-Protein Expression System
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抄録
Cytidine monophosphate (CMP) N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) is an essential enzyme for N-glycolylneuraminic acid (Neu5Gc) synthesis. In humans, Neu5Gc cannot be synthesized because of a deletion in the CMAH gene. Since Neu5Gc research has not been actively performed in comparison with Neu5Ac research, little is known about the function of Neu5Gc. Possible reasons are that CMAH for controlling Neu5Gc synthesis is not understood well at the molecular level, that commercial Neu5Gc is expensive, and that addition of exogenous Neu5Gc to glycoconjugates is not a general method because of the difficulty in obtaining CMAH. One solution to these problems is to achieve large-scale production of CMAH with enzymatic activity. To produce and purify CMAH as simply as possible, we generated simian CMAH as a secretory protein with a histidine tag using a baculovirus protein expression system. After culture of baculovirus-infected cells in serum-free medium, secretory simian CMAH (approximately 180 µg) was highly purified from the supernatant (150 mL) of cell culture. HPLC analysis showed conversion of CMP-Neu5Ac to CMP-Neu5Gc by the secretory CMAH. We succeeded in producing secretory CMAH with enzymatic activity that is easy to purify. In addition, peptide-N-glycosidase F treatment of CMAH indicated that secretory CMAH was a glycoprotein with N-glycan. It will also contribute to research on Neu5Gc function by easy-to-use methods for controlling Neu5Gc synthesis, for exogenous addition of Neu5Gc to glycoconjugates and by application to industrial Neu5Gc synthesis.
収録刊行物
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- Biological & Pharmaceutical Bulletin
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Biological & Pharmaceutical Bulletin 38 (8), 1220-1226, 2015
公益社団法人 日本薬学会
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詳細情報 詳細情報について
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- CRID
- 1390001204632113408
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- NII論文ID
- 130005090642
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- NII書誌ID
- AA10885497
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- ISSN
- 13475215
- 09186158
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- NDL書誌ID
- 026617353
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- PubMed
- 26235586
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
- Crossref
- PubMed
- CiNii Articles
- KAKEN
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- 抄録ライセンスフラグ
- 使用不可