title: Genetic engineering approach to develop next-generation reagents for endotoxin quantification.
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.
authors.affiliation: 1 LAL Research and Development Department, Seikagaku Corporation, Tokyo, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Mizumura
authors.affiliation: 1 LAL Research and Development Department, Seikagaku Corporation, Tokyo, Japan.
authors.forename: Norihiko
authors.initials: N
authors.lastname: Ogura
authors.affiliation: 2 LAL Marketing Group, Seikagaku Corporation, Tokyo, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Aketagawa
authors.affiliation: 2 LAL Marketing Group, Seikagaku Corporation, Tokyo, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Aizawa
authors.affiliation: 1 LAL Research and Development Department, Seikagaku Corporation, Tokyo, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: 3 Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
authors.forename: Shun-Ichiro
authors.initials: SI
authors.lastname: Kawabata
authors.affiliation: 1 LAL Research and Development Department, Seikagaku Corporation, Tokyo, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Oda
mesh_terms: D000818:Animals; D002457:Cell Extracts; D011415:Complement Factor B / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000032:analysis*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D006737:Horseshoe Crabs; D007202:Indicators and Reagents; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D008033:Limulus Test / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D004731:Endotoxins; D004792:Enzyme Precursors; D007202:Indicators and Reagents; D019476:Insect Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D011415:Complement Factor B; C014775:pro-clotting enzyme
keywords: Amoebocyte lysate reagent; endotoxin; horseshoe crab; mammalian cell; recombinant cascade reagent