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

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)28-31
Number of pages4
JournalJournal of Biotechnology
Volume297
DOIs
Publication statusPublished - May 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

Cite this

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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.",
author = "Patmawati and Kosuke Minamihata and Tsuneyuki Tatsuke and Man Lee and Takahiro Kusakabe and Noriho Kamiya",
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AU - Patmawati,

AU - Minamihata, Kosuke

AU - Tatsuke, Tsuneyuki

AU - Lee, Man

AU - Kusakabe, Takahiro

AU - Kamiya, Noriho

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