Recombinant Factor C and method for producing the same, and method for measuring endotoxin

1. A horseshoe crab Factor C protein having activity of Factor C,

wherein the horseshoe crab is selected from the group consisting of Tachypleus tridentatus , and Limulus polyphemus , and wherein the horseshoe crab Factor C protein is produced through being recombinantly expressed from a Chinese Hamster Ovary (CHO) DG44 cell or Human Embryonic Kidney (HEK) 293 cell, and

wherein the Factor C protein contains (α-2,3)-linked terminal sialic acid in a greater amount, as compared with a corresponding native horseshoe crab Factor C and/or a corresponding Factor C protein recombinantly expressed using Sf9 as a host cell.

2. A Factor C protein having activity of Factor C,

wherein the Factor C protein is produced through being recombinantly expressed from a Chinese Hamster Ovary (CHO) DG44 cell or Human Embryonic Kidney (HEK) 293 cell, and

wherein the expressed Factor C protein comprises an amino acid sequence selected from the group consisting of:

(A) the amino acid sequence of SEQ ID NO: 2, and the Factor C protein contains (α-2,3)-linked terminal sialic acid in a greater amount, as compared with a native horseshoe crab Factor C of Tachypleus tridentatus and/or a corresponding Factor C protein recombinantly expressed using Sf9 as a host cell; and

(B) an amino acid sequence having 90% or higher sequence identity to the amino acid sequence of SEQ ID NO:2, and having activity of Factor C, and the Factor C protein contains (α-2,3)-linked terminal sialic acid in a greater amount, as compared with a native horseshoe crab Factor C of Tachypleus tridentatus and/or a corresponding Factor C protein recombinantly expressed using Sf9 as a host cell; and

wherein the Factor C protein exhibits a residual activity of at least one selected from the group consisting of:

(1) 10% or higher in the presence of 21 mM sodium citrate; and

(2) 15% or higher in the presence of 16 mM magnesium sulfate.

3. The Factor C protein according to claim 2 , which is water soluble.

4. The Factor C protein according to claim 2 , which has a molecular weight of 115 kDa to 140 kDa as measured by SDS-PAGE under non-reducing condition.

5. The Factor C protein according to claim 4 , which has a molecular weight of 127 kDa±5 kDa.

6. The Factor C protein according to claim 2 , which exhibits a residual Factor C activity of 35% or higher in the presence of 52 mM sodium hydrogen carbonate.

7. The Factor C protein according to claim 2 , which exhibits a residual Factor C activity of 35% or higher in the presence of 214 mM sodium chloride.

8. The Factor C protein according to claim 2 , which exhibits a residual Factor C activity of 25% or higher in the presence of 16 mM magnesium sulfate.

9. The Factor C protein according to claim 2 , which exhibits a residual Factor C activity of 35% or higher in the presence of 2.5 mM calcium chloride.

10. A method for producing the Factor C protein of claim 2 , the method comprising culturing a Chinese Hamster Ovary (CHO) DG44 cell or Human Embryonic Kidney (HEK) 293 cell recombinantly expressing said Factor C protein under conditions suitable for the expression of the Factor C protein to produce the Factor C protein.

11. An endotoxin assay kit comprising the Factor C protein according to claim 2 and one or more elements.

12. A method for measuring endotoxin in a test specimen, the method comprising

a step of mixing the Factor C protein according to claim 2 , Factor B, Pro-clotting enzyme, and t-butoxycarbonyl-leucyl-glycyl-arginyl-pNA (Boc-Leu-Gly-Arg-pNA), and the test specimen, and

a step of measuring the release of pNA, thereby measuring endotoxin in the test specimen.

13. The method according to claim 12 , wherein the test specimen comprises at least one of:

(A) an ion;

(B) a cation;

(C) a metal ion;

(D) an alkali metal ion or an alkaline earth metal ion; and

(E) one or more kinds of ions selected from the group consisting of sodium ion, potassium ion, calcium ion, and magnesium ion.

14. The method according to claim 13 , wherein the test specimen comprises a cation and the final concentration of the cation after mixing the test specimen with the assay agent is 1 mM or higher.

15. The method according to claim 12 , wherein the test specimen is an injection.

16. The Factor C protein according to claim 2 , which exhibits a residual activity of 20% or higher in the presence of 21 mM sodium citrate.

17. The Factor C protein according to claim 2 , which exhibits a residual activity of 25% or higher in the presence of 52 mM sodium hydrogen carbonate.

18. The Factor C protein according to claim 2 , which exhibits a residual activity of 25% or higher in the presence of 214 mM sodium chloride.

19. The Factor C protein according to claim 1 , wherein the Factor C protein contains (α-2,3)-linked terminal sialic acid in a greater amount, as compared with a corresponding native horseshoe crab Factor C and a corresponding Factor C protein recombinantly expressed using Sf9 as a host cell.

20. The Factor C protein according to claim 2 , wherein the Factor C protein contains (α-2,3)-linked terminal sialic acid in a greater amount, as compared with a corresponding native horseshoe crab Factor C and a corresponding Factor C protein recombinantly expressed using Sf9 as a host cell.

21. A horseshoe crab Factor C protein having activity of Factor C,

wherein the horseshoe crab is Limulus polyphemus , and wherein the horseshoe crab Factor C protein is produced through being recombinantly expressed from a Human Embryonic Kidney (HEK) 293 cell, and

wherein the Factor C protein exhibits a residual activity of at least one selected from the group consisting of:

(1) 10% or higher in the presence of 21 mM sodium citrate; and

(2) 15% or higher in the presence of 16 mM magnesium sulfate.