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 language | English |
|---|---|
| Pages (from-to) | 28-31 |
| Number of pages | 4 |
| Journal | Journal of Biotechnology |
| Volume | 297 |
| DOIs | |
| Publication status | Published - May 20 2019 |
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All Science Journal Classification (ASJC) codes
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology
Cite this
Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes. / Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Man; Kusakabe, Takahiro; Kamiya, Noriho.
In: Journal of Biotechnology, Vol. 297, 20.05.2019, p. 28-31.Research output: Contribution to journal › Article
}
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, Man
AU - Kusakabe, Takahiro
AU - Kamiya, Noriho
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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UR - http://www.scopus.com/inward/citedby.url?scp=85063645660&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2019.03.007
DO - 10.1016/j.jbiotec.2019.03.007
M3 - Article
C2 - 30885655
AN - SCOPUS:85063645660
VL - 297
SP - 28
EP - 31
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
ER -