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

wherein the horseshoe crab is selected from the group consisting of Tachypleus tridentatus, Limulus polyphemus , and Carcinoscoryius rotundicauda , and wherein the horseshoe crab Factor C protein is produced through being recombinantly expressed from a Chinese Hamster Ovary (CHO) DG44 cell.

2. The Factor C protein according to claim 1 , which exhibits a residual Factor C activity of 10% or higher in the presence of 21 mM sodium citrate.

3. The Factor C protein according to claim 1 , which has a molecular weight of 115 kDa to 140 kDa as measured by SDS-PAGE under non-reducing conditions.

4. A Factor C protein having activity of Factor C, 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 protein;

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

(A) the amino acid sequence of SEQ ID NO: 2;

(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;

(C) the amino acid sequence of SEQ ID NO: 4; and

(D) an amino acid sequence having 90% or higher sequence identity to the amino acid sequence of SEQ ID NO:4, and having activity of Factor C,

wherein the Factor C protein is produced through being recombinantly expressed from a CHO DG44 cell, and

wherein the Factor C protein exhibits a residual activity of 10% or higher in the presence of 21 mM sodium citrate.

5. The Factor C protein according to claim 1 , which is water soluble.

6. A method for producing the horseshoe crab Factor C protein of claim 1 , the method comprising culturing a CHO DG44 cell recombinantly expressing said horseshoe crab Factor C protein under conditions suitable for the expression of the horseshoe crab Factor C to produce the horseshoe crab Factor C protein.

7. The method according to claim 6 , wherein the 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

(B) the amino acid sequence of SEQ ID NO: 4.

8. An endotoxin assay kit comprising the Factor C protein according to claim 1 and one or more elements.

9. A method for measuring endotoxin in a test specimen, the method comprising a step of mixing the horseshoe crab Factor C protein according to claim 1 , 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.

10. The method according to claim 9 , 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.

11. The method according to claim 10 , 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.

12. The method according to claim 9 , wherein the test specimen is an injection.

13. The Factor C protein according to claim 3 , which has a molecular weight of 127 kDa±5 kDa.

14. The Factor C protein according to claim 1 , which exhibits a residual Factor C activity of 25% or higher in the presence of 52 mM sodium hydrogen carbonate.

15. The Factor C protein according to claim 1 , which exhibits a residual Factor C activity of 25% or higher in the presence of 214 mM sodium chloride.

16. The Factor C protein according to claim 1 , which exhibits a residual Factor C activity of 15% or higher in the presence of 16 mM magnesium sulfate.

17. The Factor C protein according to claim 1 , which exhibits a residual Factor C activity of 35% or higher in the presence of 2.5 mM calcium chloride.

18. A horseshoe crab Factor C protein having activity of Factor C, wherein the horseshoe crab 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 protein and/or a horseshoe crab Factor C protein recombinantly expressed using Sf9 as a host cell,

wherein the horseshoe crab 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

(B) the amino acid sequence of SEQ ID NO: 4,

wherein the horseshoe crab Factor C protein is produced through being recombinantly expressed from a CHO DG44 cell, and

wherein the horseshoe crab Factor C protein exhibits a residual activity of 10% or higher in the presence of 21 mM sodium citrate.

19. An endotoxin assay kit comprising the Factor C protein according to claim 4 and one or more elements.

20. An endotoxin assay kit comprising the horseshoe crab Factor C protein according to claim 18 and one or more elements.