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

• Published in: Journal of biotechnology 2019-05, Vol.297, p.28-31
• Main Authors: Patmawati, Minamihata, Kosuke, Tatsuke, Tsuneyuki, Lee, Jae Man, Kusakabe, Takahiro, Kamiya, Noriho
• Format: Article
• Language: English
• Subjects: Baculovirus; Diagnostic tool; Horseradish peroxidase; Silkworm larva; Streptavidin
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2019.03.007

•Recombinant horseradish peroxidase–streptavidin fusion protein was firstly reported.•The fusion proteins were expressed in silkworm larva as soluble apo form.•Simple incubation with hemin overnight at 4 °C yielded the activated holo form.•The activated fusion protein works as a probe in enzyme-linked immunosorbent assay.•Baculovirus-silkworm expression system is promising to design Stav-fused enzymes. 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.