Expression and Activation of Horseradish Peroxidase–Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes

Patmawati xxxx, Kosuke Minamihata, Tsuneyuki Tatsuke, Man Lee, Takahiro Kusakabe, Noriho Kamiya

研究成果: ジャーナルへの寄稿記事

3 引用 (Scopus)

抄録

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.

元の言語英語
記事番号1700624
ジャーナルBiotechnology Journal
13
発行部数6
DOI
出版物ステータス出版済み - 6 1 2018

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Armoracia
Bombyx
Horseradish Peroxidase
Larva
Proteins
Enzyme-Linked Immunosorbent Assay

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Molecular Medicine

これを引用

Expression and Activation of Horseradish Peroxidase–Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes. / xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Man; Kusakabe, Takahiro; Kamiya, Noriho.

:: Biotechnology Journal, 巻 13, 番号 6, 1700624, 01.06.2018.

研究成果: ジャーナルへの寄稿記事

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