Characterization of a recombinant Bacteroides fragilis sialidase expressed in Escherichia coli

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URI http://shark.lib.kagawa-u.ac.jp/kuir/metadata/28573
Title
Characterization of a recombinant Bacteroides fragilis sialidase expressed in Escherichia coli
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Description

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)

Author
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
Publication Title
Anaerobe
Publication Title Alternative
Anaerobe.
Volume
50
Start Page
69
End Page
75
Publisher
Anaerobe Society of the Americas
Japanese Accociation for Anaerobic Infection Research
Elsevier
Published Date
2018-02-09
ISSN
1075-9964
NCID
AA11101129
PMID
29432848
DOI
10.1016/j.anaerobe.2018.02.003
Resource Type
Thesis or Dissertation
Language
eng
Resource URL
出版社版DOIリンク(URL): https://doi.org/10.1016/j.anaerobe.2018.02.003
Relation(isVersionOf)
https://doi.org/10.1016/j.anaerobe.2018.02.003
Rights
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.
Text Version
ETD
Grant ID
博甲第702号
Grant Date
2018-12-26
Degree Name
博士(医学)
Grantor
香川大学
Set
香川大学
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