Characterization of a recombinant Bacteroides fragilis sialidase expressed in Escherichia coli

( 最大 2000 件 )
URI http://shark.lib.kagawa-u.ac.jp/kuir/metadata/28573
タイトル
Characterization of a recombinant Bacteroides fragilis sialidase expressed in Escherichia coli
ファイル
内容記述

Abstract

The human gut commensal Bacteroides fragilis produces sialidases that remove a terminal sialic acid from host-derived polysaccharides. Sialidase is considered to be involved in B. fragilis infection pathology. A native B. fragilis sialidase has been purified and characterized, and was shown to be post-translationally modified by glycosylation. However, the biochemical properties of recombinant B. fragilis sialidase expressed in a heterologous host remain uncharacterized. In this study, we examined the enzymatic properties of the 60-kDa sialidase NanH1 of B. fragilis YCH46, which was prepared as a recombinant protein (rNanH1) in Escherichia coli. In E. coli rNanH1 was expressed as inclusion bodies, which were separated from soluble proteins to allow solubilization of insoluble rNanH1 in a buffer containing 8 M urea and renaturation in refolding buffer containing 100 mM CaCl2 and 50 mM L-arginine. The specific activity of renatured rNanH1 measured using 4-methylumberiferyl-α-D-N-acetyl neuraminic acid as a substrate was 6.16 μmol/min/mg. The optimal pH of rNanH1 ranged from 5.0 to 5.5. The specific activity of rNanH1 was enhanced in the presence of calcium ions. rNanH1 preferentially hydrolyzed the sialyl α2,8 linkage and cleaved sialic acids from mucin and serum proteins (e.g., fetuin and transferrin) but not from α1-acid glycoprotein, which is similar to the previously observed biochemical properties for a native sialidase purified from B. fragilis SBT3182. The results and methods described in this study will be useful for preparing and characterizing recombinant proteins for other B. fragilis sialidase isoenzymes.

KEYWORDS:

Bacteroides fragilis; Mucin; Sialic acid; Sialidase; Sialyl α2,8 linkage

(医博甲702)

著者
著者 山本 高成
著者(ヨミ) ヤマモト タカアキ
著者(別表記) Yamamoto Takaaki
掲載誌
Anaerobe
掲載誌(別表記)
Anaerobe.
50
開始ページ
69
終了ページ
75
出版者
Anaerobe Society of the Americas
Japanese Accociation for Anaerobic Infection Research
Elsevier
出版年月日
2018-02-09
ISSN
1075-9964
NCID
AA11101129
PMID
29432848
DOI
10.1016/j.anaerobe.2018.02.003
資料タイプ
学位論文
言語
英語
関連情報URL
出版社版DOIリンク(URL): https://doi.org/10.1016/j.anaerobe.2018.02.003
関連情報(isVersionOf)
https://doi.org/10.1016/j.anaerobe.2018.02.003
権利関係
エルゼビア・ジャパンより、博士論文については、機関リポジトリでの公開可能確認済み。/Elsevier Japan has confirmed the publication possibility in the institutional repository about the doctoral thesis.
博士論文(全文を含む)
学位授与番号
博甲第702号
学位授与年月日
2018-12-26
学位名
博士(医学)
学位授与機関
香川大学
区分
香川大学
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