TY - JOUR
T1 - Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes
AU - Patmawati,
AU - Minamihata, Kosuke
AU - Tatsuke, Tsuneyuki
AU - Lee, Jae Man
AU - Kusakabe, Takahiro
AU - Kamiya, Noriho
N1 - Funding Information:
This work was supported by the Japan Science and Technology Agency (JST) for the Program for Creating Start-ups from Advanced Research and Technology (START Program) and the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number JP16H04581 (to N. K.). We thank James Allen, DPhil, from Edanz Group ( www.edanzediting.com/ac ) for editing a draft of this manuscript.
Publisher Copyright:
© 2019 Elsevier B.V.
PY - 2019/5/20
Y1 - 2019/5/20
N2 - 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.
AB - 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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U2 - 10.1016/j.jbiotec.2019.03.007
DO - 10.1016/j.jbiotec.2019.03.007
M3 - Article
C2 - 30885655
AN - SCOPUS:85063645660
SN - 0168-1656
VL - 297
SP - 28
EP - 31
JO - Journal of Biotechnology
JF - Journal of Biotechnology
ER -