Recombinant factor C and method for producing the same, and method for measuring endotoxin
To provide a method for producing a horseshoe crab recombinant Factor C. The horseshoe crab recombinant Factor C is produced through expression thereof by use of mammalian cells such as CHO DG44 and HEK293 as host cells.
1. A method for imparting a salt ion resistance for a horseshoe crab Factor C, the method comprising:
recombinantly expressing and secreting the horseshoe crab Factor C into a culture broth or a culture supernatant using a mammalian cell as a host cell, and
recovering the recombinant horseshoe crab Factor C secreted from the mammalian cell,
wherein the horseshoe crab Factor C recombinantly expressed and secreted using the mammalian cell contains (α-2,3)-linked terminal sialic acid in a greater amount, as compared with a corresponding horseshoe crab Factor C expressed using a Sf9 insect cell as a host cell,
wherein the mammalian cell is a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) cell, and
wherein the horseshoe crab Factor C recombinantly expressed and secreted into a culture broth or a culture supernatant using the mammalian cell is a protein selected from the following (A), (B), (C), or (D):
(A) a protein comprising the amino acid sequence of Factor C of Tachypleus tridentatus , wherein the protein has no peptide added to the N-terminus of Factor C of Tachypleus tridentatus , and wherein the protein has activity of Factor C;
(B) a protein comprising the amino acid sequence of Factor C of Limulus polyphemus , wherein the protein has no peptide added to the N-terminus of Factor C of Limulus polyphemus , and wherein the protein has activity of Factor C;
(C) a protein comprising the amino acid sequence of Factor C of Carcinoscorpius rotundicauda , wherein the protein has no peptide added to the N-terminus of Factor C of Carcinoscorpius rotundicauda , and wherein the protein has activity of Factor C;
(D) a protein comprising the amino acid sequence of Factor C of Tachypleus gigas , wherein the protein has no peptide added to the N-terminus of Factor C of Tachypleus gigas , and wherein the protein has activity of Factor C.
2. The method according to claim 1 , wherein the salt ion is selected from the group consisting of sodium ion, potassium ion, calcium ion, and magnesium ion.
3. The method according to claim 1 , wherein the horseshoe crab Factor C recombinantly expressed and secreted using a mammalian cell exhibits a higher residual activity in the presence of 21 mM sodium citrate as compared with a corresponding horseshoe crab Factor C expressed in a Sf9 insect cell as a host cell.
4. The method according to claim 1 , wherein the horseshoe crab Factor C recombinantly expressed and secreted using a mammalian cell exhibits a higher residual activity in the presence of 52 mM sodium hydrogencarbonate as compared with a corresponding horseshoe crab Factor C expressed in a Sf9 insect cell as a host cell.
5. The method according to claim 1 , wherein the horseshoe crab Factor C recombinantly expressed and secreted using a mammalian cell exhibits a higher residual activity in the presence of 214 mM sodium chloride as compared with a corresponding horseshoe crab Factor C expressed in a Sf9 insect cell as a host cell.
6. The method according to claim 1 , wherein the horseshoe crab Factor C recombinantly expressed and secreted using a mammalian cell exhibits a higher residual activity in the presence of 16 mM magnesium sulfate as compared with a corresponding horseshoe crab Factor C expressed in a Sf9 insect cell as a host cell.
7. The method according to claim 1 , wherein the protein comprises the amino acid sequence of Factor C of Tachypleus tridentatus, Limuluspolyphemus , or Carcinoscorpius rotundicauda.
8. A method for producing an endotoxin assay agent comprising a horseshoe crab Factor C, the method comprising:
recombinantly expressing and secreting the horseshoe crab Factor C into a culture broth or a culture supernatant using a mammalian cell as a host cell,
recovering the recombinant horseshoe crab Factor C secreted from the mammalian cell, and
producing an endotoxin assay agent comprising the recombinant horseshoe crab Factor C,
wherein the horseshoe crab Factor C recombinantly expressed and secreted using the mammalian cell contains (α-2,3)-linked terminal sialic acid in a greater amount, as compared with a corresponding horseshoe crab Factor C expressed using a Sf9 insect cell as a host cell,
wherein the mammalian cell is a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) cell, and
wherein the horseshoe crab Factor C recombinantly expressed and secreted into a culture broth or a culture supernatant using the mammalian cell is a protein selected from the following (A), (B), (C), or (D):
(A) a protein comprising the amino acid sequence of Factor C of Tachypleus tridentatus , wherein the protein has no peptide added to the N-terminus of Factor C of Tachypleus tridentatus , and wherein the protein has activity of Factor C;
(B) a protein comprising the amino acid sequence of Factor C of Limulus polyphemus , wherein the protein has no peptide added to the N-terminus of Factor C of Limulus polyphemus , and wherein the protein has activity of Factor C;
(C) a protein comprising the amino acid sequence of Factor C of Carcinoscorpius rotundicauda , wherein the protein has no peptide added to the N-terminus of Factor C of Carcinoscorpius rotundicauda , and wherein the protein has activity of Factor C;
(D) a protein comprising the amino acid sequence of Factor C of Tachypleus gigas , wherein the protein has no peptide added to the N-terminus of Factor C of Tachypleus gigas , and wherein the protein has activity of Factor C.
9. The method according to claim 8 , wherein the horseshoe crab Factor C recombinantly expressed and secreted using a mammalian cell exhibits a higher residual activity in the presence of 21 mM sodium citrate as compared with a corresponding horseshoe crab Factor C expressed in a Sf9 insect cell as a host cell.
10. The method according to claim 8 , wherein the horseshoe crab Factor C recombinantly expressed and secreted using a mammalian cell exhibits a higher residual activity in the presence of 52 mM sodium hydrogencarbonate as compared with a corresponding horseshoe crab Factor C expressed in a Sf9 insect cell as a host cell.
11. The method according to claim 8 , wherein the horseshoe crab Factor C recombinantly expressed and secreted using a mammalian cell exhibits a higher residual activity in the presence of 214 mM sodium chloride as compared with a corresponding horseshoe crab Factor C expressed in a Sf9 insect cell as a host cell.
12. The method according to claim 8 , wherein the horseshoe crab Factor C recombinantly expressed and secreted using a mammalian cell exhibits a higher residual activity in the presence of 16 mM magnesium sulfate as compared with a corresponding horseshoe crab Factor C expressed in a Sf9 insect cell as a host cell.
13. The method according to claim 8 , wherein the protein comprises the amino acid sequence of Factor C of Tachypleus tridentatus, Limuluspolyphemus , or Carcinoscorpius rotundicauda.