Method for producing factor C recombinant protein and endotoxin-measuring agent
AssigneeSeikagaku Corporation
InventorsHikaru Mizumura, Maki Aizawa, Toshio Oda
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Abstract
A method for rapidly and highly sensitively measuring endotoxin relies on an endotoxin-measuring agent, which includes a factor C derived from Tachypleus tridentatus that does not have His-tag sequence at the C-terminus, a factor B of a horseshoe crab, and a proclotting enzyme of a horseshoe crab. Each of these proteins can be a recombinant protein obtainable by being expressed using a stably expressing cell line of an insect cell as a host.
Claims10
A method for producing an endotoxin measuring agent, comprising:
- (a) incorporating each of DNAs (1) to (3) below into three vectors respectively, with the proviso that the vector is not a virus:
- (1) a DNA encoding a factor C, which DNA is DNA (A′) or (B′) below, and which factor C does not have His-tag sequence or any peptide attached at the C terminus:
- (A′) a DNA having a sequence consisting of the nucleotide sequence shown in SEQ ID NO:1;
- (B′) a DNA encoding a protein having a sequence consisting of an amino acid sequence having an identity of 90% or higher to the amino acid sequence shown in SEQ ID NO:2, wherein the protein has factor C activity greater than the activity of natural factor C under the same condition;
- (2) a DNA encoding a factor B of a horseshoe crab;
- and
- (3) a DNA encoding a proclotting enzyme of a horseshoe crab;
- (b) introducing the three vectors, into which said each DNA was incorporated, into insect cells to incorporate said each DNA into a chromosome of the insect cells;
- (c) allowing the insect cells, into which said each DNA was incorporated, to express the protein encoded by said each DNA;
- (d) recovering a solution comprising the expressed proteins;
- and
- (e) formulating the endotoxin measuring agent comprising the expressed proteins.
A method for producing a factor C recombinant protein for use in an endotoxin measuring agent, comprising:
- (a) incorporating a DNA encoding the factor C recombinant protein into a vector, wherein the factor C recombinant protein encoded by the DNA is a protein (A) or (B) below, and does not have His-tag sequence or any peptide attached at the C-terminus, with the proviso that the vector is not a virus:
- (A) a protein consisting of an amino acid sequence shown in SEQ ID NO:2;
- (B) a protein consisting of an amino acid sequence having an identity of 90% or higher to the amino acid sequence shown in SEQ ID NO:2, wherein the protein has factor C activity greater than the activity of natural factor C under the same condition;
- (b) introducing the vector, into which said DNA was incorporated, into insect cells to incorporate said DNA into a chromosome of the insect cells;
- (c) allowing the insect cells, into which said DNA was incorporated, to express the factor C recombinant protein encoded by said DNA;
- and
- (d) recovering a solution containing the expressed protein.
Dependencies are inferred from the claim text.