Recombinant Factor C and method for producing the same, and method for measuring endotoxin
A horseshoe crab Factor C protein having activity of Factor C, wherein the horseshoe crab is selected from Tachypleus tridentatus, Limulus polyphemus , and Carcinoscorpius rotundicauda , and wherein the horseshoe crab Factor C protein is produced through being recombinantly expressed from a Chinese Hamster Ovary (CHO) DG44 cell or HEK cell.
1. A method for producing an endotoxin assay agent comprising a horseshoe crab Factor C, a horseshoe crab Factor B, and a horseshoe crab Pro-clotting enzyme, the method comprising:
expressing the horseshoe crab Factor C in a Chinese Hamster Ovary cell or a Human Embryonic Kidney cell as a host cell, secreting the horseshoe crab Factor C into an extracellular culture supernatant, and recovering the horseshoe crab Factor C in the culture supernatant, wherein the recovered horseshoe Factor C has activity of Factor C, and wherein the horseshoe crab Factor C is a Tachypleus tridentatus Factor C or a Limulus polyphemus Factor C,
expressing the horseshoe crab Factor B having activity of Factor B in a mammalian cell as a host cell,
expressing the horseshoe crab Pro-clotting enzyme in a mammalian cell as a host cell, and
producing an endotoxin assay agent comprising the horseshoe crab Factor C, the horseshoe crab Factor B, and the horseshoe crab Pro-clotting enzyme,
wherein the host cell for expressing the horseshoe crab Factor C, the host cell for expressing the horseshoe crab Factor B, and the host cell for expressing the horseshoe crab Pro-clotting enzyme may be the same or different.
2. The method according to claim 1 , wherein the mammalian cell expressing the Factor B is a Chinese Hamster Ovary cell or a Human Embryonic Kidney cell.
3. The method according to claim 1 , wherein the mammalian cell expressing the Pro-clotting enzyme is a Chinese Hamster Ovary cell or a Human Embryonic Kidney cell.
4. The method according to claim 1 , wherein the horseshoe crab for the Factor B and/or the horseshoe crab for the Pro-clotting enzyme is selected from the group consisting of Tachypleus tridentatus and Limulus polyphemus.
5. The method according to claim 1 , wherein the horseshoe crab Factor C contains (α-2,3)-linked terminal sialic acid in a greater amount, as compared with a corresponding Factor C protein recombinantly expressed using Sf9 as a host cell.
6. The method according to claim 1 , wherein the horseshoe crab Factor C has a molecular weight of 115 kDa to 130 kDa, as measured through SDS-PAGE under a nonreducing condition.
7. The method according to claim 1 , further comprising filtering the culture supernatant comprising the horseshoe crab Factor C.
8. The method according to claim 1 , wherein the endotoxin assay agent is formulated as a solid, a liquid, or a gel.
9. The method according to claim 1 , wherein the horseshoe crab Factor C, the horseshoe crab Factor B, and the horseshoe crab Pro-clotting enzyme are separately expressed in individually established host cells.
10. A method for measuring endotoxin in a test specimen, the method comprising
expressing a horseshoe crab Factor C in a Chinese Hamster Ovary cell or a Human Embryonic Kidney cell as a host cell, secreting the horseshoe crab Factor C into an extracellular culture supernatant, and recovering the horseshoe crab Factor C in the culture supernatant, wherein the recovered horseshoe Factor C has activity of Factor C, and wherein the horseshoe crab Factor C is a Tachypleus tridentatus Factor C or a Limulus polyphemus Factor C,
expressing a horseshoe crab Factor B having activity of Factor B in a mammalian cell as a host cell,
expressing a horseshoe crab Pro-clotting enzyme in a mammalian cell as a host cell,
producing an endotoxin assay agent comprising the horseshoe crab Factor C, the horseshoe crab Factor B, and the horseshoe crab Pro-clotting enzyme,
mixing the endotoxin assay agent and a test specimen, and
measuring progress of a cascade reaction,
wherein the host cell for expressing the horseshoe crab Factor C, the host cell for expressing the horseshoe crab Factor B, and the host cell for expressing the horseshoe crab Pro-clotting enzyme may be the same or different.