Genetic engineering approach to develop next-generation reagents for endotoxin quantification

Hikaru Mizumura, Norihiko Ogura, Jun Aketagawa, Maki Aizawa, Yuki Kobayashi, Shun-Ichiro Kawabata, Toshio Oda

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.

Original languageEnglish
Pages (from-to)136-146
Number of pages11
JournalInnate Immunity
Volume23
Issue number2
DOIs
Publication statusPublished - Jan 1 2017

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Genetic Engineering
Endotoxins
Horseshoe Crabs
Insects
Peptide Hydrolases
Genetic Techniques
Enzyme Precursors
Complement Factor B
Glycosylation
Injections

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Molecular Biology
  • Cell Biology
  • Infectious Diseases

Cite this

Genetic engineering approach to develop next-generation reagents for endotoxin quantification. / Mizumura, Hikaru; Ogura, Norihiko; Aketagawa, Jun; Aizawa, Maki; Kobayashi, Yuki; Kawabata, Shun-Ichiro; Oda, Toshio.

In: Innate Immunity, Vol. 23, No. 2, 01.01.2017, p. 136-146.

Research output: Contribution to journalArticle

Mizumura, H, Ogura, N, Aketagawa, J, Aizawa, M, Kobayashi, Y, Kawabata, S-I & Oda, T 2017, 'Genetic engineering approach to develop next-generation reagents for endotoxin quantification', Innate Immunity, vol. 23, no. 2, pp. 136-146. https://doi.org/10.1177/1753425916681074
Mizumura, Hikaru ; Ogura, Norihiko ; Aketagawa, Jun ; Aizawa, Maki ; Kobayashi, Yuki ; Kawabata, Shun-Ichiro ; Oda, Toshio. / Genetic engineering approach to develop next-generation reagents for endotoxin quantification. In: Innate Immunity. 2017 ; Vol. 23, No. 2. pp. 136-146.
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