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

• Published in: Innate immunity (London, England) Vol. 23; no. 2; p. 136
• Main Authors: Mizumura, Hikaru, Ogura, Norihiko, Aketagawa, Jun, Aizawa, Maki, Kobayashi, Yuki, Kawabata, Shun-Ichiro, Oda, Toshio
• Format: Journal Article
• Language: English
• Published: United States 01-02-2017
• Subjects: Animals; Cell Extracts; Complement Factor B - genetics; Complement Factor B - metabolism; Endopeptidases - genetics; Endopeptidases - metabolism; Endotoxins - analysis; Enzyme Precursors - genetics; Enzyme Precursors - metabolism; Genetic Engineering; Horseshoe Crabs; Indicators and Reagents; Insect Proteins - genetics; Insect Proteins - metabolism; Limulus Test - methods; Recombinant Proteins - genetics; Reference Standards; Sensitivity and Specificity; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; horseshoe crab; mammalian cell; Amoebocyte lysate reagent; recombinant cascade reagent; endotoxin
• ISSN: 1753-4267
• DOI: 10.1177/1753425916681074

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.