Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes

Patmawati, 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

抄録

Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP) 4 –Stav or Stav–(HRP) 4 , respectively) using a baculovirus-silkworm expression system. Both (HRP) 4 –Stav and Stav–(HRP) 4 were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP) 4 –Stav and Stav–(HRP) 4 could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP) 4 –Stav was twofold higher than that of Stav–(HRP) 4 , and the sensitivity of (HRP) 4 -Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP) 4 –Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.

元の言語英語
ページ(範囲)28-31
ページ数4
ジャーナルJournal of Biotechnology
297
DOI
出版物ステータス出版済み - 5 20 2019

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Armoracia
Bombyx
Baculoviridae
Streptavidin
Horseradish Peroxidase
Enzymes
Assays
Proteins
Immunosorbents
Enzyme-Linked Immunosorbent Assay
Antigens
Antibodies
Amplification
Ovalbumin
Fusion reactions
Recombinant Fusion Proteins
Molecular Probes
Hemin

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

これを引用

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abstract = "Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP) 4 –Stav or Stav–(HRP) 4 , respectively) using a baculovirus-silkworm expression system. Both (HRP) 4 –Stav and Stav–(HRP) 4 were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP) 4 –Stav and Stav–(HRP) 4 could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP) 4 –Stav was twofold higher than that of Stav–(HRP) 4 , and the sensitivity of (HRP) 4 -Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP) 4 –Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.",
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AU - Lee, Man

AU - Kusakabe, Takahiro

AU - Kamiya, Noriho

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