# US 10,144,923 B2 · Recombinant Factor C and method for producing the same, and method for measuring endotoxin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,144,923 B2 - Document ID: US10144923B2 - Status: Granted - Kind: B2 - Application number: 14/650,767 - Filed: 2013-12-10 - Published: 2015-10-29 - Granted: 2018-12-04 - Earliest claimed: 2012-12-10 - Assignees: Seikagaku Corporation - Applicants: Seikagaku Corporation - Inventors: Hikaru Mizumura; Toshio Oda; Shun-ichiro Kawabata - Web page: https://patentgrep.com/patent/US10144923B2 ## CPC - C12N 9/6408: {Serine endopeptidases (3.4.21)} - C12Y 304/21069: Protein C activated (3.4.21.69) - C12Y 304/21084: limulus clotting factor C (3.4.21.84) - G01N 33/56911: {Bacteria} - G01N 33/579: involving limulus lysate - G01N 2333/96411: Serine endopeptidases (3.4.21) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) 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. ### Claim 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. ### Claim 5 (depends on 1) The Factor C protein according to claim 1, which is water soluble. ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 6) 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. ### Claim 8 (depends on 1) An endotoxin assay kit comprising the Factor C protein according to claim 1 and one or more elements. ### Claim 9 (depends on 1) 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. ### Claim 10 (depends on 9) 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. ### Claim 11 (depends on 10) 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. ### Claim 12 (depends on 9) The method according to claim 9, wherein the test specimen is an injection. ### Claim 13 (depends on 3) The Factor C protein according to claim 3, which has a molecular weight of 127 kDa±5 kDa. ### Claim 14 (depends on 1) 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. ### Claim 15 (depends on 1) 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. ### Claim 16 (depends on 1) 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. ### Claim 17 (depends on 1) 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. ### Claim 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. ### Claim 19 (depends on 4) An endotoxin assay kit comprising the Factor C protein according to claim 4 and one or more elements. ### Claim 20 (depends on 18) An endotoxin assay kit comprising the horseshoe crab Factor C protein according to claim 18 and one or more elements.