WO2024204498A1 - 組換えタンパク質 - Google Patents

組換えタンパク質 Download PDF

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Publication number
WO2024204498A1
WO2024204498A1 PCT/JP2024/012572 JP2024012572W WO2024204498A1 WO 2024204498 A1 WO2024204498 A1 WO 2024204498A1 JP 2024012572 W JP2024012572 W JP 2024012572W WO 2024204498 A1 WO2024204498 A1 WO 2024204498A1
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Prior art keywords
factor
amino acid
protein
seq
endotoxin
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French (fr)
Japanese (ja)
Inventor
悠子 一柳
隼也 石川
敦 一柳
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Kikkoman Corp
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Kikkoman Corp
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Priority to EP24780599.7A priority patent/EP4692354A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21084—Serine endopeptidases (3.4.21) limulus clotting factor C (3.4.21.84)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS

Definitions

  • the present invention relates to a recombinant protein. More specifically, the present invention relates to a recombinant protein for measuring, for example, endotoxin.
  • Endotoxin is a lipopolysaccharide that constitutes the cell wall of gram-negative bacteria. Since endotoxin is a very stable substance, it is difficult to remove or inactivate if it is mixed into pharmaceuticals, medical devices, dialysis fluids, etc. during the manufacturing or use process. Endotoxin is a typical pyrogen, and it is known that even a very small amount (ng or pg order) of endotoxin-contaminated material can cause fever in the person or animal who receives it and can cause diseases with extremely high fatality rates, such as intravascular blood coagulation associated with sepsis.
  • the Limulus test is the mainstream method for measuring endotoxin.
  • the Limulus test is a test method based on the phenomenon in which an extract of horseshoe crab blood cells (Limulus Amebocyte Lysate, hereafter also referred to as LAL) reacts with endotoxin, and known methods include the gelation method, turbidimetric method, colorimetric method, and fluorescent method (see, for example, Patent Documents 1 and 2).
  • Measurement reagents that use LAL are referred to as LAL reagents, lysate reagents, Limulus reagents, etc., all of which are synonymous.
  • Endotoxin converts the enzyme precursor Factor C to activated Factor C.
  • Activated Factor C acts as a hydrolase and converts the enzyme precursor Factor B to activated Factor B.
  • Activated Factor B is also a hydrolase and converts the clotting enzyme precursor (proclotting enzyme) to activated clotting enzyme (clotting enzyme).
  • Clotting enzymes are hydrolases. This hydrolase activity is used to hydrolyze synthetic substrates such as peptide-labeled compounds, and the products or disappearances are measured by turbidimetry, chromogenic substrate method, fluorescent substrate method, luminescent substrate method, etc.
  • Horseshoe crab genera include Limulus, Tachypleus, and Carcinoscorpius. Species include Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda. Horseshoe crab hemocyte extracts are a finite biological resource. On the other hand, the demand for LAL reagents is increasing, and continuing to rely on horseshoe crab hemocyte extracts as a source of LAL reagents is not desirable from the standpoint of sustainability.
  • Patent document 3 describes a recombinant protein derived from Limulus and the DNA encoding it.
  • Patent documents 4 to 11 describe recombinant proteins.
  • Non-Patent Document 1 describes recombinant Factor C, a synthetic substitute for horseshoe crab hemocyte extracts for endotoxin detection, aimed at preserving horseshoe crabs.
  • Recombinant Factor C is commercially available, but the authors of Non-Patent Document 1 point out that the lack of progress in switching from LAL reagents to recombinant Factor C is due to the fact that the disclosed recombinant Factor C is protected by an exclusive patent, and the pharmaceutical industry has been reluctant to rely on a single supplier for endotoxin detection reagents. Thus, the fact that recombinant Factor C is already known does not make other options unnecessary.
  • the present disclosure aims to provide a recombinant Factor C protein that is thermostable and at least partially solves the problems of the prior art.
  • the inventors have surprisingly found that, as an example, the thermal stability of Factor C is improved by introducing a specific mutation into wild-type Factor C having the amino acid sequence of SEQ ID NO: 3, and have completed the present invention, which includes this as one embodiment.
  • the present disclosure encompasses the following embodiments.
  • the Factor C protein before modification has an amino acid sequence identity of 95% or more with SEQ ID NO: 3;
  • the modified Factor C protein has a substitution of Leu at the position corresponding to F191 of SEQ ID NO: 3; Substitution of Leu at the position corresponding to F423; Substitution of Leu at the position corresponding to F558, Substitution of the position corresponding to F670 with Tyr or Leu; Substitution of Leu at the position corresponding to F828; Substitution of Ala or Val at the position corresponding to L971; a substitution at the position corresponding to A974 with Leu, and/or a substitution at the position corresponding to G1004 with Ser or Thr,
  • a modified Factor C protein which has improved thermal stability as compared with the unmodified Factor C protein.
  • a method for producing a Factor C protein comprising culturing the cells described in embodiment 4 and obtaining the Factor C protein described in embodiment 1.
  • a method for producing an endotoxin detection reagent comprising culturing the cells described in embodiment 4 and obtaining the Factor C protein described in embodiment 1.
  • a method for detecting endotoxin comprising the steps of contacting a sample with the protein described in embodiment 1 or the reagent described in embodiment 3, and detecting endotoxin.
  • the effect of the present invention is that hydrolytic enzymes can be obtained without relying on horseshoe crab hemocyte extracts.
  • the present disclosure provides a recombinant Factor C protein with improved thermal stability having at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% amino acid sequence identity to SEQ ID NO:3 and having a predetermined amino acid substitution.
  • the present disclosure provides a recombinant Factor C protein having an amino acid sequence in which a specific amino acid substitution has been introduced into SEQ ID NO:3, and one or several amino acids have been substituted, deleted, or added at a position other than the specific position.
  • “one or several" with respect to the substitution, deletion, or addition of amino acids may be 1 to 20, for example, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, for example, 1 or 2.
  • each position of the sequence is defined based on SEQ ID NO: 3 as the reference sequence.
  • SEQ ID NO: 3 is the wild-type Factor C protein derived from Limulus polyphemus.
  • wild type refers to the trait that is most commonly present in nature within a homogeneous population.
  • the correspondence of amino acid positions can be easily identified by comparing the amino acid sequences of various Factor Cs using, for example, existing amino acid homology analysis software, such as GENETYX (manufactured by GENETYX).
  • GENETYX existing amino acid homology analysis software
  • the amino acid position of Factor C corresponding to position X of the amino acid sequence of SEQ ID NO: 3 can be identified by aligning the amino acid sequence of Factor C with the amino acid sequence of SEQ ID NO: 3.
  • a mutation may be artificially introduced into Factor C of SEQ ID NO:3. This can be done by artificially introducing a mutation into the sequence of the gene encoding the recombinant Factor C.
  • Mutations may be introduced artificially with the intention of achieving some specific effect, or may be introduced randomly or non-artificially. Examples of mutations introduced with the intention of achieving a specific effect include addition, deletion, or modification of a sequence to increase the expression level of Factor C, addition, deletion, or modification of a sequence to improve the purification efficiency of Factor C protein, and various mutations that impart practically desirable properties to Factor C protein.
  • the identity of amino acid sequences and gene sequences can be calculated using programs such as maximum matching and search homology in GENETYX (GENETYX), multiple alignment in CLUSTAL W, pairwise alignment by BLAST, and other programs.
  • GENETYX GENETYX
  • CLUSTAL W CLUSTAL W
  • pairwise alignment by BLAST pairwise alignment by BLAST
  • amino acid sequence identity when two or more Factor Cs are aligned, the positions of amino acids that are identical in the two or more Factor Cs can be examined. Based on this information, identical regions in the amino acid sequences can be determined.
  • the percent identity refers to the percentage calculated by aligning two or more amino acid sequences using BLAST (BLASTP) or the like for amino acid sequences, with the total number of amino acids in the aligned regions as the denominator and the number of positions occupied by identical amino acids as the numerator. Therefore, normally, when two or more amino acid sequences have a region where no identity is found at all, for example when one of the amino acid sequences has an additional sequence at the C-terminus where no identity is found at all, the region with no identity cannot be aligned and is therefore not used in calculating the percent identity.
  • BLASTP BLAST
  • amino acids that are similar in two or more Factor Cs can be aligned using CLUSTALW, in which case the algorithm Blosum62 is used, and amino acids that are determined to be similar when multiple amino acid sequences are aligned are sometimes called similar amino acids.
  • amino acid substitutions may be due to substitutions between such similar amino acids.
  • alignments can be used to examine regions of identical amino acid sequences and positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, homologous regions (also called highly conserved regions) in the amino acid sequences can be determined.
  • Factor C can also be subjected to high-throughput screening to obtain functional recombinant Factor C mutants.
  • a library of transformed or transduced strains carrying mutated Factor C genes can be prepared and subjected to microtiter plate-based high-throughput screening or droplet microfluidic-based ultra-high-throughput screening. Examples include constructing a combinatorial library of mutant genes encoding variants and then screening a large population of mutant Factor C using phage display (e.g., Chem. Rev. 105 (11): 4056-72, 2005), yeast display (e.g., Comb Chem High Throughput Screen.
  • phage display e.g., Chem. Rev. 105 (11): 4056-72, 2005
  • yeast display e.g., Comb Chem High Throughput Screen.
  • Libraries may be used to transform suitable cells, such as electrocompetent EBY-100 cells, to obtain approximately 10 million mutants.
  • Yeast cells transformed with the libraries may then be subjected to cell sorting.
  • Polydimethoxylsiloxane (PDMS) microfluidic devices fabricated using standard soft lithography techniques may also be used.
  • Flow focus devices may be used to form monodisperse droplets.
  • the droplets formed containing the individual mutants may be subjected to a suitable sorting device.
  • the presence or absence of Factor C activity may be used to select cells. For example, a reaction solution whose absorbance changes when Factor C acts on the cells may be used.
  • a 96-well plate, a 192-well plate, a 384-well plate, a 9600-well plate, or the like and a plate reader may be used to measure the change in absorbance. Detection may be performed using a luminescence system, a color system, or a fluorescence system instead of an absorption system. Mutation introduction and selection may be repeated multiple times. The mutations referred to here include substitutions, insertions, deletions, and/or additions of amino acids.
  • 1 to 10 mutations can be introduced into Factor C such as SEQ ID NO: 3, and activity can be confirmed.
  • 1 to 10 further mutations can be introduced, and their activity can be confirmed.
  • a series of high-throughput screening (for example, the above-mentioned method of obtaining and screening about 10 million mutants) can be repeated for 2 or more rounds, 5 or more rounds, 10 or more rounds, 15 or more rounds, 20 or more rounds, 30 or more rounds, 40 or more rounds, for example, 50 or more rounds.
  • Mutations may be introduced at one or more positions from the first amino acid to the last amino acid in the full-length amino acid sequence of Factor C.
  • regions important for the function of the enzyme such as the active center, substrate recognition site, endotoxin recognition site, and their vicinity, are excluded.
  • Factor C is widely used in industry, and those skilled in the art are familiar with the regions important for the function of the enzyme, including the active center, substrate recognition site, and endotoxin recognition site.
  • one or more mutations may be introduced first at positions 1 to 10 of the full-length sequence of Factor C. Next, starting from a recombinant Factor C mutant confirmed to have activity, one or more mutations may be further introduced at positions 11 to 20, and activity may be confirmed.
  • n times may be repeated n times (n ⁇ 102).
  • one or more mutations may be introduced at positions 1011 to 1020.
  • regions important for the function of the enzyme or regions not intended to be modified may be skipped as appropriate. For example, position 967 of SEQ ID NO: 3 or a position corresponding thereto can be skipped.
  • no mutations that eliminate the activity of Factor C are introduced.
  • positions corresponding to positions 967, 810, 866, 26, 61, 62, and 63 of SEQ ID NO: 3 are not mutated.
  • positions 1 to 25 of SEQ ID NO: 3 can be deleted.
  • Mutations may be introduced randomly or by rational design.
  • the mutations introduced by rational design or randomly may be conservative amino acid substitutions.
  • Conservative amino acid substitutions include amino acid substitutions in which the amino acid before and after the substitution have similar chemical properties (e.g., Stryer et al., Biochemistry, 5th ed., 2002, pp. 44-49).
  • conservative amino acid substitutions may be selected from the group consisting of (i) a basic amino acid with a different kind of basic amino acid; (ii) an acidic amino acid with a different kind of acidic amino acid; (iii) an aromatic amino acid with a different kind of aromatic amino acid; (iv) a non-polar aliphatic amino acid with a different kind of non-polar aliphatic amino acid; and (v) a polar uncharged amino acid with a different kind of polar uncharged amino acid.
  • the basic amino acid may be selected from, for example, arginine, histidine, and lysine.
  • the acidic amino acid may be, for example, aspartic acid or glutamic acid.
  • Aromatic amino acids may be selected from, for example, phenylalanine, tyrosine, and tryptophan.
  • Nonpolar aliphatic amino acids may be selected from, for example, glycine, alanine, valine, leucine, methionine, proline, and isoleucine.
  • Polar uncharged amino acids may be selected from, for example, serine, threonine, cysteine, asparagine, and glutamine.
  • Conservative amino acid substitutions are highly likely to maintain the tertiary structure and have activity because the chemical properties of the amino acid residue before and after substitution are similar, and the position at which the conservative amino acid substitution is made is the same position in the tertiary structure of the protein.
  • the mutations introduced by rational design or randomly include substitutions with functionally similar amino acids.
  • Tables of functionally similar amino acids are widely known in the art.
  • the substitutions with functionally similar amino acids may be of any of the following amino acid classes: 1) Glycine (G), Alanine (A); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (N), lysine (K), histidine (H); 5) Isoleucine (I), Leucine (L), Valine (V), Proline (P); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), Methionine (M).
  • non-conservative amino acid substitutions are substitutions of an amino acid with any amino acid that does not fall within the conservative substitutions (i) to (v) outlined above.
  • the amino acid substitution may be a non-conservative amino acid substitution. In this case, for example, it may be confirmed whether protease activity is maintained before and after the non-conservative amino acid substitution is introduced. If activity is confirmed, the non-conservative amino acid substitution may be adopted.
  • the amino acid substitution may be a substitution with a similar amino acid (similarity substitution).
  • a substitution with a similar amino acid refers to a substitution with an amino acid that is evaluated as a positive value or a neutral value (zero) in the amino acid substitution matrix used in the ClustalW software and the Blosum62 algorithm (see, e.g., S. Heinkoff and J. G. Henikoff, Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 10915-10919, 1992, especially FIG. 2 therein, and Thompson, Nucleic Acid Research, 1994, Vol. 22, No. 22, pp. 4673-4680).
  • the matrix table was generated from aligned sequence segments of approximately 2000 blocks of over 500 related proteins. Moreover, whether starting from a single matrix or using a subset of proteins, repeated application leads to roughly the same set of scores. Therefore, the substitution matrix is considered versatile. It is the most widely used approach, taking advantage of the fact that homologous sequences are evolutionarily related. Therefore, variants with similar substitutions in a Factor C are more likely to be active.
  • the conservative amino acid substitution or the substitution with a functionally similar amino acid is not present in an area important to the function of the enzyme, such as the active center, substrate recognition site, coenzyme recognition motif, or the vicinity thereof, and therefore does not significantly affect the activity of the enzyme.
  • the conservative amino acid substitution or the substitution with a functionally similar amino acid is present in the active center, substrate recognition site, coenzyme recognition motif, or the vicinity thereof, but does not substantially affect the activity of the enzyme.
  • the recombinant Factor C may contain amino acid deletions compared to the sequence prior to the amino acid substitution.
  • the amino acid deletions are not in regions critical to the function of the enzyme and therefore do not significantly affect the activity of the enzyme.
  • the deletions may be short deletions of 1-2 amino acids.
  • the amino acid sequence of one Factor C may be compared to the amino acid sequence of another Factor C, and if an amino acid is deleted in one sequence, the deletion may be introduced into the other Factor C. As both Factor Cs exhibit activity, such deletions are likely not to significantly affect the activity of the enzyme.
  • Recombinant Factor C may also include those in which additional amino acids have been inserted compared to the pre-mutation sequence.
  • the amino acid insertion is not in an area critical to the function of the enzyme, such as the active center, substrate recognition site, coenzyme recognition motif, or adjacent thereto, and therefore does not significantly affect the activity of the enzyme.
  • the insertion may be 1-4 amino acids.
  • the amino acid sequence of one Factor C may be compared to the amino acid sequence of another Factor C, and if an amino acid has been inserted in one sequence, the inserted amino acid may be introduced into the other Factor C. Since both Factor Cs exhibit activity, such an insertion is likely not to significantly affect the activity of the enzyme.
  • Recombinant Factor C may also include those in which additional amino acids have been added compared to the pre-mutation sequence.
  • the amino acid additions are made to the N-terminus or C-terminus of Factor C and do not significantly affect the activity of the enzyme.
  • the additions may be 1-6 amino acids, 1-5 amino acids, e.g., 1-4 amino acids.
  • Examples of additions include, but are not limited to, short stretches of histidine residues (e.g., 2-6 histidine residues) to aid in purification of Factor C.
  • additions also include, but are not limited to, the addition of a signal peptide to aid in expression of Factor C. Signal peptides include known signal sequences or functional equivalents thereof.
  • Mutations may be introduced into Factor C so as not to destroy secondary structures or structural motifs, such as alpha helix structures or beta sheet structures. Regions of secondary structure may be identified, for example, by secondary structure prediction algorithms. Examples of prediction algorithms include, but are not limited to, NetSurfP-2.0. The same applies to other structural motifs, such as nests and niches. From the N-terminus to the C-terminus, Factor C has an EGF-like domain, three Sushi domains, one LCCL domain, one C-type lectin domain, two further Sushi domains, and a trypsin domain. Mutations may be introduced into Factor C so as not to destroy these domains or higher-order structures.
  • amino acid residues or amino acid sequence motifs essential for Factor C activity are not substituted. In certain embodiments, conservative amino acid substitutions or similar substitutions can be made at these positions, but the activity of the variants after the substitutions is confirmed.
  • amino acids are not deleted or inserted before or after the amino acid residues essential for Factor C activity.
  • the positions before or after the amino acid residues essential for Factor C activity refer to positions one or two positions N-terminal or one or two positions C-terminal of the amino acid residue essential for the activity.
  • amino acids can be deleted or inserted before or after the amino acid residues essential for Factor C activity, but the activity of the variants after the substitutions is confirmed. Whether a variant has activity can be routinely confirmed, for example, by high-throughput screening.
  • Factor C has a catalytic triad consisting of serine, histidine, and aspartic acid (or glutamic acid).
  • serine at position 967, the histidine at position 810, and the aspartic acid at position 866 constitute the catalytic triad.
  • the amino terminal amino acid is involved in endotoxin recognition.
  • the arginine at position 1 is involved in endotoxin recognition.
  • Factor C has an arginine-tryptophan-arginine motif (RWR), which is a motif for endotoxin recognition.
  • RWR arginine-tryptophan-arginine motif
  • the arginine at position 61, the tryptophan at position 62, and the arginine at position 63 constitute the motif for endotoxin recognition.
  • these positions are not mutated.
  • these positions can be mutated, in which case the activity of the mutated variant is confirmed.
  • the amino terminal amino acid may be lysine or arginine.
  • the arginine-tryptophan-arginine motif RWR
  • one or both of the arginines may be replaced with lysine
  • the tryptophan may be replaced with tyrosine or phenylalanine.
  • Positions 1 to 25 of Factor C are removed in the mature protein and are considered to be a signal sequence, which is cleaved off during the production process of Factor C.
  • the signal sequence is composed of the methionine at position 1 to the serine at position 25.
  • the recombinant Factor C of the present disclosure has Factor C activity.
  • the presence or absence of Factor C activity can be measured, for example, according to the method described in the Examples.
  • polynucleotides in an embodiment, provides a polynucleotide encoding recombinant Factor C (hereinafter also referred to as "recombinant Factor C gene").
  • the sequence of the polynucleotide can be easily determined based on the amino acid sequence of recombinant Factor C.
  • a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 includes, but is not limited to, the polynucleotide of SEQ ID NO: 4.
  • the polynucleotide encoding the recombinant Factor C is, for example, (i) a nucleotide sequence having a sequence identity of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more over the entire length to the nucleotide sequence of SEQ ID NO: 4, and encoding an active Factor C; (ii) a nucleotide sequence encoding an active Factor C, in which one or several nucleotides are substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 4; or (iii) a nucleotide sequence selected from the group consisting of SEQ ID NO: 4; may include.
  • chromosomal DNA or mRNA can be extracted from tissues or cells of horseshoe crabs capable of producing Factor C by standard methods.
  • cDNA can be synthesized using the mRNA as a template. The chromosomal DNA or cDNA obtained in this way can be used to prepare a chromosomal DNA or cDNA library.
  • a suitable probe DNA is synthesized based on the amino acid sequence of Factor C, and a polynucleotide encoding Factor C is selected from a chromosomal DNA or cDNA library using the probe DNA.
  • a suitable primer DNA is prepared based on the amino acid sequence, and a suitable polymerase chain reaction (PCR) such as 5'RACE or 3'RACE is used to amplify DNA containing the desired nucleotide fragment encoding Factor C, and these DNA fragments are linked to obtain DNA containing the full length of nucleotides encoding the desired Factor C.
  • PCR polymerase chain reaction
  • nucleotide sequence encoding Factor C may be artificially synthesized.
  • Such an artificial gene synthesis service is provided, for example, by Integrated DNA Technologies.
  • the Factor C gene can be mutated by any known method according to the intended form of mutation, including a method of contacting the Factor C gene or a recombinant DNA incorporating the gene with a mutagenic agent, an ultraviolet irradiation method, a genetic engineering method, or a method making full use of a protein engineering method.
  • mutagenic agents used in the above mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil.
  • the conditions for this contact and action can be varied according to the type of drug used, and are not particularly limited as long as the desired mutation can actually be induced in the Factor C gene.
  • the desired mutation can be induced by contacting and acting for 10 minutes or more, preferably 10 to 180 minutes, at a drug concentration of preferably 0.5 to 12 M, at a reaction temperature of 20 to 80°C.
  • ultraviolet irradiation it can also be performed according to the usual method as described above.
  • a method that utilizes protein engineering techniques is generally known as Site-Specific Mutagenesis.
  • the desired modified Factor C gene can also be directly synthesized by organic synthesis or enzymatic synthesis.
  • the base sequence of the Factor C gene can be confirmed, for example, using a multi-capillary DNA analysis system such as the Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific).
  • the present disclosure relates to a vector comprising the polynucleotide.
  • these Factor C genes are linked to various vectors according to standard methods.
  • examples of vectors include plasmids, but any other vectors known to those skilled in the art, such as bacteriophages and cosmids, can also be used.
  • the type of vector can be selected depending on the host cell, and specifically, for example, pET16-b or pKK223-3, etc. are preferred.
  • the present disclosure relates to a host cell comprising the polynucleotide or vector described above.
  • the host cell may be, but is not limited to, a bacterium such as E. coli or Bacillus subtilis, a yeast cell, an insect cell, an animal cell (e.g., a mammalian cell), or a plant cell, preferably a bacterial cell such as E. coli.
  • the Factor C gene obtained as described above can be incorporated into a vector such as a bacteriophage, cosmid, or a plasmid used for transformation of prokaryotic or eukaryotic cells by a conventional method, and a host corresponding to each vector can be transformed by a conventional method.
  • a microorganism belonging to the genus Escherichia for example, the obtained recombinant DNA can be used as a host to transform or transduce, for example, E. coli K-12 strain, or E. coli B strain, preferably E. coli JM109 strain, E. coli DH5 ⁇ strain, E. coli BL21 strain, E.
  • coli BL21 (DE3) strain etc.
  • a method for transferring a recombinant vector into such a host cell for example, when the host cell is a microorganism belonging to Escherichia coli, a method of transferring a recombinant DNA in the presence of calcium ions can be adopted. Furthermore, electroporation may be used.
  • the Factor C gene may be codon-optimized according to the expression host.
  • the present disclosure relates to a method for producing recombinant Factor C, comprising the step of culturing the host cell.
  • the culture can be carried out by various known methods, and may be a solid culture method, but is preferably a liquid culture method.
  • the production method may include a step of culturing the host cell under conditions that allow expression of the recombinant Factor C protein, and optionally a step of isolating the recombinant Factor C from the culture or culture solution.
  • the condition that allows expression of the recombinant Factor C protein means that the Factor C gene is transcribed and translated, and a polypeptide encoded by the gene is produced.
  • the medium for culturing the host cells may be one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran infusion, to which one or more inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and carbohydrate raw materials, vitamins, etc. may also be appropriately added as necessary.
  • nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran infusion
  • inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and carbohydrate raw materials, vitamins, etc. may also be appropriately added as necessary.
  • mammalian cells When mammalian cells are used as host cells, media commonly used for culturing mammalian cells can be used, such as DMEM medium (Sigma), RPMI1640 medium (Sigma), ExpiCHOTM Expression Medium (Thermo Fisher Scientific), etc. Mammalian cells can be cultured, for example, at 35° C. to 38° C., e.g., 36° C. to 38° C., under 5% to 8% CO2 supply, by static culture or suspension culture.
  • DMEM medium Sigma
  • RPMI1640 medium Sigma
  • ExpiCHOTM Expression Medium Thermo Fisher Scientific
  • media commonly used for culturing insect cells may be used, such as Sf-900TM medium (Thermo Fisher), Sf-900TM II medium (Thermo Fisher), Sf-900TM III medium (Thermo Fisher), GibcoTM TC-100 insect medium (Thermo Fisher), Schneider insect medium (Sigma-Aldrich), Grace insect medium (Sigma-Aldrich), TNM-FH insect medium (Sigma-Aldrich), Express FiveTM SFM medium (Thermo Fisher), etc.
  • Sf-900TM medium Thermo Fisher
  • Sf-900TM II medium Thermo Fisher
  • Sf-900TM III medium Thermo Fisher
  • GibcoTM TC-100 insect medium Thermo Fisher
  • Schneider insect medium Sigma-Aldrich
  • Grace insect medium Sigma-Aldrich
  • TNM-FH insect medium Sigma-Aldrich
  • Express FiveTM SFM medium Thermo Fisher
  • insect cells examples include, but are not limited to, those commonly used for expressing recombinant proteins, such as IPLB-Sf9, IPLB-Sf21, IPLB-SF+, High-FiveTM, Schneider S2, BmN, etc.
  • Known cell lines such as cell lines derived from the silkworm (Bombyx mori), armyworm moth (Mamestra brassicae), fall armyworm moth (Spodoptera frugiperda), nettle moth (Trichoplusia ni), and fruit fly (Drosophila melanogaster), such as Drosophila embryo-derived cell lines and silkworm embryo-derived cell lines, may also be used.
  • the conditions for culturing insect cells are not particularly limited, and conditions normally used for culturing insect cells can be used. Furthermore, the normally used conditions may be modified as appropriate.
  • the culture can be performed, for example, at 25 to 30°C, e.g., 26 to 29°C, e.g., 27°C to 28°C, by shaking culture or stationary culture.
  • the method for expressing a protein using an insect cell as a host is not particularly limited, and any method commonly used for expressing recombinant proteins may be used.
  • an insect cell may be infected with a virus incorporating a gene encoding a target protein.
  • Viruses used for transformation of insect cells include known viruses, such as baculoviruses, such as nucleopolyhedroviruses (NPVs), such as AcNPV (Autographa californica NPV) and BmNPV (Bombix mori NPV).
  • Nucleic acid may be introduced into a virus by a conventional method, for example, by homologous recombination using a transfer vector.
  • transfer vectors examples include pPSC8 (Protein Sciences), pVL1393 (Pharmingen), and pFastBac (Invitrogen).
  • a vector incorporating a gene encoding a target protein can be introduced into an insect cell, and the gene can be incorporated into the host chromosome to express the recombinant protein.
  • vectors include, but are not limited to, any known insect vector, such as the pAc series, pVL series, pIZ series, pIZ/V5-His vector (Thermo Fisher), and pIZT/V5-His (Thermo Fisher).
  • the initial pH of the medium is suitably adjusted to pH 7-9.
  • the culture is preferably performed at a culture temperature of 20-42°C, preferably at a culture temperature of around 25-37°C for 4-24 hours, more preferably at a culture temperature of around 25-37°C for 8-16 hours, by aeration and stirring submerged culture, shaking culture, static culture, etc.
  • yeast include yeasts belonging to the genera Zygosaccharomyces, Saccharomyces, Pichia, and Candida.
  • fungi include the genera Aspergillus and Tricoderma.
  • plant cells include plant cells transformed with Agrobacterium. Mammalian cells include CHO, HEK293, etc.
  • Factor C can be collected from the culture by using a conventional enzyme collection method.
  • eukaryotic cells such as yeast or fungi
  • the cells can be ultrasonically disrupted, ground, or the enzyme can be extracted using a lytic enzyme such as lysozyme, or the cells can be shaken or left to lyse in the presence of toluene or the like to excrete the enzyme from the cells.
  • the solution can then be filtered, centrifuged, or the like to remove solids, and nucleic acids can be removed using streptomycin sulfate, protamine sulfate, manganese sulfate, or the like as necessary.
  • Ammonium sulfate, alcohol, acetone, or the like can then be added to fractionate the solution, and the precipitate can be collected to obtain the crude Factor C enzyme.
  • the culture liquid, culture supernatant, or a cell disruption extract can be obtained by, for example, disrupting the cells using a conventional method to obtain a cell disruption extract, and nucleic acids can be removed or ammonium sulfate precipitation can be performed as necessary to obtain the crude Factor C enzyme.
  • a purified Factor C enzyme from the above-mentioned crude Factor C enzyme, for example, gel filtration using Sephadex, Superdex, Ultrogel, etc., adsorption elution using ion exchange carriers, hydrophobic carriers, hydroxyapatite, electrophoresis using polyacrylamide gel, etc., precipitation methods such as sucrose density gradient centrifugation, affinity chromatography, fractionation using molecular sieve membranes or hollow fiber membranes, etc. can be appropriately selected or combined to obtain a purified Factor C enzyme. In this way, the desired Factor C can be obtained.
  • Factor C may be subjected to a glycosylation treatment or may be used as it is without being subjected to the treatment.
  • the Factor C produced can be used in the endotoxin detection reagent or method for detecting endotoxin described in this specification. If the Factor C produced has relatively high heat resistance, such an enzyme will have relatively improved shelf life, and can contribute to the creation of detection reagents and detection kits that are superior to conventional Factor C in terms of distribution and storage.
  • the present disclosure provides an endotoxin detection reagent comprising recombinant Factor C.
  • the endotoxin detection reagent may contain other components typically contained in conventional reagents, such as a buffer, a stabilizer, and a synthetic substrate, in addition to recombinant Factor C.
  • the form of the reagent is not particularly limited, and examples thereof include a form of a lyophilized powder, a liquid reagent, and the like.
  • Factor C When endotoxin is present in the sample, Factor C is converted to activated Factor C. Activated Factor C can hydrolyze the labeled peptide substrate. The hydrolyzed label can be detected by turbidimetry, chromogenic substrate method, fluorescent substrate method, luminescent substrate method, etc. This allows endotoxin in the sample to be detected.
  • the endotoxin may be any type that is recognized as a substrate by the Factor C used, and may be natural or chemically synthesized. Any known endotoxin derivative may also be used.
  • the present disclosure provides an endotoxin detection kit.
  • the endotoxin detection kit may include an endotoxin detection reagent, and optionally an instruction manual, a standard substance, a syringe, a needle, a sampling device for a specimen, etc.
  • the present disclosure relates to a method for detecting endotoxin, comprising using recombinant Factor C.
  • the method may comprise contacting a sample with recombinant Factor C and detecting a label generated from a synthetic substrate.
  • recombinant Factor C having the sequence of SEQ ID NO:3 may have amino acid substitutions. Positions at which amino acids may be substituted include, but are not limited to, positions corresponding to 423, 558, 670, 971, 191, 828, 974, and 1004 of SEQ ID NO:3.
  • the recombinant Factor C may have the following amino acid substitutions, but the substituted amino acids are not limited to these.
  • a mutant having any one of the following mutations may be referred to as a single mutant Mon.
  • the recombinant Factor C may have two or more, three or more, four or more, five or more, six or more, seven or more, for example eight, of the above amino acid substitutions.
  • the recombinant Factor C may have the following amino acid substitutions.
  • the amino acids in parentheses may be any amino acid.
  • “L971(A,V)” means that the position corresponding to position 971 of SEQ ID NO:3 may be Ala or Val.
  • the recombinant Factor C may have the following amino acid substitutions: The amino acids in brackets mean that they can be any amino acid.
  • Collection S-2 For convenience, the above collection of mutants is referred to as collection S-2.
  • S-2 ⁇ Mon-2, Dou-2, Tri-2, Qua-2, Pen-2, Hex-2, Hep-2, Oct-2, Non-2 ⁇
  • the recombinant Factor C may have any combination of mutations in sets S-1 and S-2. For convenience, such mutants are referred to as set S-3. In one embodiment, the recombinant Factor C mutant may have one or more reversion mutations starting from a recombinant Factor C mutant included in sets S-1, S-2, or S-3 (however, the number of reversion mutations does not exceed the number of amino acid substitutions introduced).
  • a reversion mutation is a mutation that introduces a mutation at a position where an amino acid substitution has been introduced, reverting the amino acid to the state before the amino acid substitution. For convenience, a collection of such reversion mutants is referred to as set S-4.
  • the recombinant Factor C mutants contained in Sets S-1, S-2, S-3, or S-4 may have improved thermal stability compared to the recombinant Factor C before mutation. These may be referred to as mutants with improved thermal stability in this specification.
  • the number of recombinant Factor C mutants contained in Sets S-1, S-2, S-3, or S-4 is limited, and a person skilled in the art can confirm their thermal stability through routine confirmation procedures.
  • the disclosure provides a recombinant Factor C protein mutant having a mutation included in set S-1, S-2, S-3, or S-4 and having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, e.g., 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more amino acid sequence identity to SEQ ID NO:3.
  • thermal stability can be evaluated, for example, by using the remaining activity when Factor C is heat-treated at a predetermined temperature for a predetermined time as an indicator.
  • the thermal stability of Factor C can be evaluated by comparing the remaining activity rate after heat-treating Factor C under high temperature conditions, for example, at temperatures of 30 to 56°C, 35 to 55°C, 40 to 50°C, for example, 42 to 48°C, for example, 45 to 48°C, for a certain period of time, for example, 5 to 120 minutes, 10 to 60 minutes, for example, 15 to 30 minutes.
  • the residual activity of Factor C is the activity after heat treatment, assuming that the Factor C activity before exposure to the above-mentioned high-temperature conditions is 1. For example, if the residual activity after heat treatment is halved, the residual activity after heat treatment will be 0.5 compared to the activity before heat treatment of 1.
  • the residual activity rate of Factor C is calculated as the ratio of the activity after heat treatment to the Factor C activity before exposure to the above-mentioned high-temperature conditions.
  • improved thermal stability refers to a case in which the residual activity of a Factor C mutant when acted under the above conditions is improved by 1.01-fold or more, 1.02-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, or 2-fold or more compared to Factor C before the introduction of the mutations disclosed herein (mutations included in Sets S-1, S-2, S-3, or S-4).
  • improved thermal stability refers to a case in which the residual activity of the Factor C mutant when acted under the above conditions shows an improvement of 1% or more, 2% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the residual activity of Factor C before the introduction of the mutation disclosed herein.
  • Factor C may have an amino acid sequence selected from the group consisting of: (i) an amino acid sequence having 95% or more, 95.5%, 96% or more, 96.5%, 97% or more, 97.5%, 98% or more, 98.5%, 99% or more, or 99.5% or more amino acid sequence identity compared to SEQ ID NO:3; (ii) the amino acid sequence of SEQ ID NO: 3; or (iii) the amino acid sequence of (i) or (ii) further comprising one or more mutations included in Sets S-1, S-2, S-3 or S-4, such as a substitution of Leu at the position corresponding to position F191 of SEQ ID NO: 3; Substitution of Leu at the position corresponding to F423; Substitution of Leu at the position corresponding to F558, Substitution of the position corresponding to F670 with Tyr or Leu; Substitution of Leu at the position corresponding to F828; Substitution of Ala or Val at the position corresponding to L97
  • the present disclosure provides a Factor C having a residual activity of more than 44% after heat treatment at 48°C for 30 minutes, e.g., 46% or more, e.g., 47% or more, e.g., 50% or more, e.g., 55% or more, e.g., 78% or more, e.g., 107% or more, e.g., 111% or more, e.g., 116% or more, e.g., 123% or more, assuming that the activity before heat treatment is 100%.
  • the present disclosure provides an endotoxin detection reagent comprising such Factor C.
  • the present disclosure provides an endotoxin detection method comprising the steps of contacting the Factor C or the reagent with a sample and detecting endotoxin.
  • the recombinant Factor C of the present disclosure excludes wild-type sequences themselves.
  • the recombinant Factor C of the present disclosure excludes natural products.
  • wild-type sequences or natural products that become known after the filing or publication of the present disclosure are also excluded.
  • the present disclosure makes it possible to obtain hydrolytic enzymes without relying on horseshoe crab hemocyte extracts.
  • the present disclosure also makes it possible to detect endotoxins without relying on horseshoe crab hemocyte extracts.
  • the present disclosure also provides an alternative method that differs from conventional recombinant Factor C.
  • Example 1 Construction of a plasmid vector for Factor C expression (1-1) Construction of a vector fragment
  • the vector used was pIZT/V5-His (Invitrogen), and 1 ng of the vector was mixed with 25 ⁇ L of KOD One PCR Master Mix (Toyobo), 1.5 ⁇ L of 5 ⁇ M forward primer (SEQ ID NO: 1), and 1.5 ⁇ L of 5 ⁇ M reverse primer (SEQ ID NO: 2), and the mixture was diluted to 50 ⁇ L with ultrapure water and subjected to PCR.
  • the PCR conditions were 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 68 ° C. for 20 seconds, and the fragment was amplified by repeating this cycle 25 times.
  • the resulting PCR product was added with 2 ⁇ L of the restriction enzyme DpnI (New England Biolabs) and incubated at 37°C for 1 hour.
  • the reaction product was purified using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel) and used as a vector fragment.
  • This insert fragment was dissolved in ultrapure water to a concentration of 50 ng/ ⁇ L, and 1.5 ⁇ L of the solution was mixed with 1 ⁇ L of the 100 ng/ ⁇ L vector fragment obtained in (1-1) and 2 ⁇ L of 5x In-Fusion Snap Assembly Master Mix (Clontech), and the solution was diluted to 10 ⁇ L with ultrapure water and reacted at 50°C for 15 minutes. 5 ⁇ L of the resulting In-Fusion reaction mixture was used to transform 50 ⁇ L of ECOS (trademark) Competent E. coli JM109 (Nippon Gene), which was then plated on LB agar medium supplemented with 25 ⁇ g/mL Zeocin and incubated overnight at 37°C to obtain transformants.
  • ECOS trademark Competent E. coli JM109
  • the colonies obtained were inoculated into 2.5 mL of LB liquid medium supplemented with 25 ⁇ g/mL Zeocin, grown overnight at 37°C at 200 rpm, and a plasmid was prepared using FastGene Plasmid Mini Kit (Nihon Genetics).
  • the base sequence was deciphered using DNA sequence analysis by FASMAC, and it was confirmed that a plasmid vector (pIZT/V5-LpFC) for expressing Factor C had been constructed.
  • the colonies whose sequences had been confirmed were inoculated into 5 mL of 2TY medium supplemented with 25 ⁇ g/mL Zeocin, grown overnight at 37°C at 200 rpm, and an endotoxin-free plasmid was prepared using NucleoBond (registered trademark) Xtra Midi EF (Macherey-Nagel).
  • the surface of the vial was sterilized with 70% ethanol, and the entire amount was transferred to the above-mentioned Erlenmeyer flask after gentle mixing.
  • the flask cap was loosened, and the cells were cultured at 28° C. with rotary shaking at 125 rpm.
  • the cell culture medium After 3 days, a portion of the cell culture medium was sampled and mixed with 4% trypan blue solution in a 1:1 ratio, and the cell number and viability were measured using a CountessTM Automated Cell Counter (manufactured by Thermo Fisher Scientific). If the cell concentration was 3 ⁇ 10 6 to 6 ⁇ 10 6 cells/mL, the cells were passaged to a concentration of 2 ⁇ 10 5 to 4 ⁇ 10 5 cells/mL. Cell passage was performed periodically once every 3 to 4 days during the implementation period of this example.
  • Cellfectin II Reagent (Thermo Fisher Scientific) was mixed with Sf9 cells in Sf-900 III SFM (150 ⁇ L) for each sample, and the two were left to stand for 15 minutes. Note that Cellfectin II Reagent was mixed well by inversion before use. The plasmid and Cellfectin II Reagent that had been left to stand for each time were mixed and added to each well containing the cells by sprinkling, and the cells were gently shaken left and right and left to stand for 96 hours at 28°C. After the end of the culture, the transfection efficiency was confirmed using a fluorescence microscope BZ-X810 (Keyence), and the supernatant was sampled. The supernatant was filtered through a syringe filter DISMIC 13CP020AS (Advantec Toyo) to obtain the LpFC culture supernatant.
  • the X-axis shows the measurement time, and the Y-axis shows the fluorescence value.
  • the value calculated by the SLOPE function was used as "endotoxin-dependent activity" in the following evaluation.
  • As a control a sample with Otsuka distilled water added instead of endotoxin was also tested at the same time, and the activity in this case was considered to be "endotoxin-independent activity.” Samples with significantly higher activity were excluded from further testing.
  • the endotoxin standard was Control Standard Endotoxin (CSE), 10 ng/vial (manufactured by CAPE COD), which was adjusted to 10 EU/mL with Otsuka distilled water, and the fluorescent substrate was Boc-Asp(OBzl)-Pro-Arg-MCA (manufactured by Peptide Institute) dissolved in CultureSure (registered trademark) DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and then adjusted to 0.25 mM with Otsuka distilled water. All reagents used were endotoxin-free or biotechnology grade.
  • CSE Control Standard Endotoxin
  • 10 ng/vial manufactured by CAPE COD
  • the fluorescent substrate was Boc-Asp(OBzl)-Pro-Arg-MCA (manufactured by Peptide Institute) dissolved in CultureSure (registered trademark) DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd
  • pIZT/V5-LpFC was diluted with ultrapure water to 40 ng/ ⁇ L, 0.5 ⁇ L of the diluted solution was mixed with 10 ⁇ L of KOD One PCR Master Mix, 1.2 ⁇ L of 5 ⁇ M Fw primer, and 1.2 ⁇ L of 5 ⁇ M Fw primer, and then the mixture was diluted to 20 ⁇ L with ultrapure water and subjected to PCR.
  • the template plasmid vector, Fw primer, and Rv primer used in PCR are shown in the table below.
  • the PCR conditions were 98°C for 10 seconds, 55°C for 5 seconds, and 68°C for 35 seconds, with one cycle consisting of 15 repetitions of this cycle to amplify the fragment.
  • the PCR product obtained was used to obtain transformants in the same manner as in Example 1, and an endotoxin-free plasmid vector for expressing mutant LpFC was prepared.
  • mutant LpFC expression plasmid vector insect cells were cultured and transient transfection was performed in the same manner as in Example 1 to obtain mutant LpFC culture supernatant. Activity evaluation was also performed in the same manner as in Example 1.
  • the recombinant protein of the present disclosure may be used as a protease for various purposes.
  • the recombinant protein of the present disclosure may be used for endotoxin measurement or endotoxin detection, but is not limited thereto.
  • SEQ ID NO: 1-2 Primers for amplifying pIZT/V5 fragment
  • SEQ ID NO: 3 Amino acid sequence of LpFC
  • SEQ ID NO: 4 Base sequence of LpFC gene
  • SEQ ID NO: 5 Homologous sequence added upstream of start codon
  • SEQ ID NO: 6 Homologous sequence added downstream of stop codon
  • SEQ ID NO: 7-25 Primers for introducing LpFC mutations
  • SEQ ID NO: 3 Amino acid sequence of LpFC
  • SEQ ID NO: 4 Nucleotide sequence of LpFC gene

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See also references of EP4692354A1
STRYER ET AL., BIOCHEMISTRY, vol. 5, 2002, pages 44 - 49
THOMPSON, NUCLEIC ACID RESEARCH, vol. 22, no. 22, 1994, pages 4673 - 4680

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