JP2009183250A - Method for preparing c2 domain protein of blood coagulation factor viii - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and efficient method of preparing a FVIII C2 protein using gene recombination technology wherein E. coli is used as a host. <P>SOLUTION: The simple and efficient method of preparing the above protein includes a process of adding urea into bacterial cells of recombinant E. coli wherein a polynucleotide including a C2 domain gene of blood coagulation factor VIII is introduced in an amount in a range of 1M to 3M relative to the intracellular soluble fraction present subsequent to the expression of the C2 domain protein of blood coagulation factor VIII, and a process of subsequently purifying the protein by performing metal chelate chromatography in the presence of urea in an amount in a range of 1M to 3M. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a human blood coagulation factor VIII C2 domain protein using genetic recombination technology using E. coli as a host, and more specifically, a bacterium after the protein is expressed in recombinant E. coli. The present invention relates to a simple and efficient method for producing the protein, characterized by adding an appropriate concentration of urea to a body soluble fraction and a buffer used for purification.

  Human blood coagulation factor VIII (FVIII) is an important protein that acts as a cofactor of FIXa in the blood coagulation system, and when it is deficient, hemophilia A develops. Statistically, one in 5,000 males is genetically deficient in this factor, and it is estimated that more than 400,000 patients worldwide need to be administered FVIII. FVIII has long been separated and purified from human blood as one of the blood products, but there is concern about the contamination of various pathogens such as HIV, HCV, and prions. Therefore, research was conducted on a method for producing FVIII using genetic engineering techniques. As a result, in the early 1990s, genetically modified products began to be used in clinical settings, and now blood products and genetically modified products are being used. Both are on the market. However, since FVIII has a low content in blood and is not highly productive even if genetic engineering techniques are used, there is a need for an improved FVIII purification method. As prior art of the FVIII purification method, affinity chromatography using a mouse monoclonal antibody as a ligand, which is also used in the production process, and affinity chromatography using a low molecular weight affinity peptide found using phage display as a ligand. (Non-Patent Documents 1 and 2), but it is not always a sufficient purification method, and there is a lot of room for technological development.

  FVIII is a large glycoprotein having a molecular weight of 280,000 to 300,000, and is composed of six domains, A1-A2-B-A3-C1-C2 (Non-patent Document 3). Of these, the C2 domain located at the C-terminus (hereinafter abbreviated as FVIIIC2) binds to von Willebrand factor (vWF) in blood and is often a target for neutralizing antibodies (inhibitors) Patent Document 4) is recognized as an important domain in the physiological blood coagulation process and also in the treatment of hemophilia A. In addition, FVIIIC2 has a region that binds to acidic cell membrane (model of activated platelet cell membrane) rich in phosphatidylserine (Non-patent document 5), and is structurally and functionally separated from other domains. (Non-patent Document 3), utilization as a target of an anticoagulant and utilization as a target for a ligand for affinity chromatography for purifying FVIII are expected. However, as described above, since FVIII has a low content in blood and is very expensive, it is economically expensive to prepare FVIIIC2 using FVIII as a raw material. Thus, research has been conducted on a method for producing FVIIIC2 using genetic engineering techniques.

  As production of FVIIIC2 using a genetic engineering technique, production using E. coli (Non-patent Document 6) or Pichia yeast (Patent Document 1, Non-patent Document 7) as a host is disclosed. Since no chain is added, it is easier to produce using E. coli than Pichia yeast. In Non-Patent Document 6 where intracellular expression of FVIIIC2 was performed using recombinant Escherichia coli, the culture temperature was lowered from 37 ° C. to 16 ° C. after addition of IPTG, a gene expression inducer, and cultured for 16 to 20 hours. A method for collecting bodies is disclosed. Although the reason why the above operation is necessary is not disclosed, it can be assumed that aggregates are produced when cultured at 37 ° C. However, since the growth of Escherichia coli is greatly suppressed at a temperature of 16 ° C., the cell density does not rise sufficiently even if cultured for a long time, and the decomposition of FVIIIC2 proceeds during the culture, so that high productivity is obtained. It is difficult.

US Published Patent Publication No. 2006/0293505 Nord K. Et al., European Journal of Biochemistry, 268, 4269-4277, 2001. Kelly B. D. Et al., Journal of Chromatography A, 1038, 121-130, 2004. Stoilova-McPhie S. Blood, 99 (4), 1215-1223, 2002. Zhong D. Blood, 92 (1), 136-142, 1998. Lewis D.D. A. Et al., Blood Coagulation and Fibrinolysis, 14, 361-368, 2003. Spigel P.M. C. Chemistry and Biology, 11, 1413-1422, 2004. Pratt K.M. P. Nature, 402, 439-442, 1999.

  Since the blood coagulation factor VIII C2 domain (FVIIIC2) has the above-mentioned utility, an economical and simple production method is desired. The present invention provides a simple and efficient method for producing the FVIIIC2 protein using gene recombination technology using E. coli as a host.

  In order to solve the above problems, in the present invention, by examining the purification conditions in detail for a blood coagulation factor VIII C2 domain (FVIIIC2) protein expressed in recombinant E. coli, the protein can be conveniently and efficiently obtained. I found a way to get it.

That is, the present invention includes the following inventions:
A first invention is a method for producing FVIIIC2 protein using a recombinant Escherichia coli into which a polynucleotide containing an FVIIIC2 gene has been introduced. In the method for producing FVIIIC2 protein after expressing FVIIIC2 protein in the recombinant Escherichia coli On the other hand, the protein is purified by adding urea in the range of 1M to 3M and then performing metal chelate chromatography in the presence of urea in the range of 1M to 3M, and thereby producing FVIIIC2 protein Is the method.

  According to the second invention, the initial concentration of imidazole is within the range of 20 to 50 mM and the final concentration is 200 to 500 mM with respect to the soluble fraction after the FVIIIC2 protein is expressed in the cells of the recombinant E. coli. The method for producing FVIIIC2 protein according to the first invention, wherein the protein is eluted from the metal chelate chromatography carrier and purified by applying a concentration gradient within the range of.

  According to a third invention, the FVIIIC2 gene according to the first or second invention is characterized in that the polynucleotide containing the FVIIIC2 gene has at least a histidine tag sequence added to the 5 ′ end side of the FVIIIC2 gene. A method for producing a protein.

  A fourth invention is the method for producing an FVIIIC2 protein according to the first or second invention, wherein the polynucleotide containing the FVIIIC2 gene is a polynucleotide consisting of SEQ ID NO: 1.

  Hereinafter, the present invention will be described in detail.

Since the FVIIIC2 gene in the present invention has a known human FVIII gene sequence (GenBank accession No. NM_019863), the gene can be prepared by chemically synthesizing a region corresponding to FVIIIC2. In addition, the gene can be more easily prepared using a commercially available human cDNA library or cDNA clone, and one example is Ultimate ^™ Human ORF CLONE manufactured by Invitrogen.

  In the present invention, the polynucleotide containing the FVIIIC2 gene refers to a polynucleotide obtained by adding a polynucleotide encoding 1 to 40 amino acids to the 5 ′ terminal side or the 3 ′ terminal side to the polynucleotide of the FVIIIC2 gene. Examples of the polynucleotide to be added to the 5 ′ end side or 3 ′ end side of the FVIIIC2 gene include polynucleotides encoding tag sequences such as histidine tag, S tag, GST tag, and intein tag. The nucleotide is a polynucleotide encoding at least a histidine tag on the 5 ′ end side of the FVIIIC2 gene in terms of the productivity of the fusion protein, the purification efficiency of the fusion protein, the stability of the fusion protein, the availability of the chromatography carrier, and the economy. Is preferably added. More preferably, the polynucleotide comprising the FVIIIC2 gene is a polynucleotide consisting of SEQ ID NO: 1, wherein a 114 base polynucleotide of the histidine tag region of pET28a (Novagen) is added to the 5 ′ end of the FVIIIC2 gene.

The recombinant Escherichia coli strain that expresses FVIIIC2 protein in the present invention is not particularly limited, and can include HB101, JM109, DH5α, C600 and derivatives thereof in the K12 strain, and BL21 and derivatives thereof in the B strain, Furthermore, it can select more finely according to the compatibility with the protein to produce or its polynucleotide. In the examples, BL21-CodonPlus ^™ (DE3) -RIPL (manufactured by Stratagene), which is a preferred example of a recombinant E. coli strain, was selected as a recombinant E. coli strain, but the present invention is not limited to this. .

There are no particular limitations on the type of medium used for culturing recombinant Escherichia coli in the present invention, and examples include LB medium that is a natural medium, 2 × YT medium, and M9 medium that is a synthetic medium. There is no particular limitation on the culture temperature until the gene expression inducer is added after inoculating the recombinant E. coli in the medium. A preferable example is 37 ° C., which is the optimal growth temperature for E. coli. The addition of the gene expression inducer is preferably carried out during the logarithmic growth phase of the recombinant Escherichia coli. D. _It is preferable to add a gene expression inducer at the time when ₆₀₀ reaches 1.0 to 1.2. The type of gene expression inducer is not particularly limited, and examples thereof include IPTG and arabinose. Regarding the culture temperature when FVIIIC2 protein is expressed in the cells of recombinant E. coli after adding the gene expression inducer, from Example 4, the majority of the expressed FVIIIC2 protein is insolubilized when cultured at 37 ° C. When cultured at 25 ° C., the expression level of FVIIIC2 protein is small, and therefore, it is preferable to culture at a temperature of 26 ° C. to 35 ° C. Further, when cultured at 29 ° C. and 32 ° C. in Example 4, the FVIIIC2 protein is mainly expressed as a soluble protein in a large amount, and therefore, it is particularly preferable to culture at 29 ° C. to 32 ° C. In addition, the buffer used when disrupting recombinant E. coli after expression of FVIIIC2 protein contains a low concentration (preferably 20 to 50 mM) of imidazole to suppress nonspecific adsorption to the column in the subsequent purification operation. Is preferably added.

  In the method for producing FVIIIC2 protein in the present invention, urea is added within the range of 1M to 3M to the soluble fraction after the FVIIIC2 protein is expressed in the cells of recombinant E. coli, and then within the range of 1M to 3M. The protein is purified by metal chelate chromatography in the presence of urea. GE Healthcare's Ni Sepharose 6 Fast Flow, Ni Sepharose High Performance, Qiagen's Ni-NTA Agarose as the metal chelate chromatography carrier, and GE Healthcare's HisTrap HP, HisTrap FF, Examples include His SpinTrap and Qiagen Ni-NTA Spin Column. The concentration of urea added to the cell-soluble fraction and the buffer used for purification is within the above-mentioned range, in which irreversible protein denaturation by urea is slight and non-specific interaction can be suppressed. It is more preferable to carry out purification by adding 2M urea. Moreover, by adding urea of the above concentration to the intracellular soluble fraction and the buffer used for purification, the effect of promoting the exposure of the histidine tag to the solvent and the effect of suppressing the action of various proteases present in the bacterial cell extract Can also be expected. In particular, a chelating agent such as EDTA, which is an inhibitor of metal protease, cannot be used in metal chelate chromatography, so that the effect of adding urea is high.

  In the present invention, in addition to the above-mentioned conditions, the FVIIIC2 protein purification further comprises adsorbing the protein on a metal chelate chromatography support, and then passing a buffer solution containing a low concentration of imidazole at least 10 times the column volume. Thus, after effectively removing non-specifically adsorbed protein, the purification method recovers the target protein by adding a high concentration of imidazole or lowering the pH of the buffer solution. In many cases, imidazole is added or the pH of the buffer solution is lowered in one step, but for more precise purification, it is preferable to elute with a gradient. However, since it is generally difficult to control the pH gradient, it is more preferable to elute by applying a gradient of imidazole. The initial concentration and the final concentration of imidazole may be set in accordance with the separation state of the target protein and the contaminating protein. The most preferable imidazole concentration in the present invention is 20 to 50 mM for the initial concentration and 200 to 500 mM for the final concentration. Although the eluted protein can be used as it is, it is preferably used after removing high-concentration urea or imidazole using dialysis or gel permeation chromatography carrier. By the above operation, the three-dimensional structure and activity of the protein weakly denatured with urea can be recovered.

  For evaluation of the FVIIIC2 protein obtained by the production method of the present invention, it is preferable to use a compound having a known binding activity, and examples thereof include antibodies and chemically synthesized peptides. Examples of antibodies that can be used for affinity purification of FVIII include those for which the binding target is the C2 domain, and polyclonal or monoclonal antibodies prepared using FVIIIC2 as an antigen. Examples of chemically synthesized peptides include the peptides listed in Non-Patent Documents 1 and 2 and analogs thereof. Examples of the method for measuring the activity of FVIIIC2 protein include surface plasmon resonance (SPR) analysis, ELISA, and isothermal titration calorimetry (ITC).

  In the production of blood coagulation factor VIII C2 domain (FVIIIC2) protein using genetic recombination technology using E. coli as a host, the soluble fraction in the cell after the protein is expressed in recombinant E. coli, and It is characterized by adding an appropriate concentration of urea to the buffer used for purification. Since the FVIIIC2 protein obtained by the production method of the present invention is an important domain in the physiological blood coagulation process and the treatment of hemophilia A, the treatment and diagnosis of coagulation diseases including hemophilia Or can be used for research.

  Examples will be shown below to describe the present invention in more detail. However, these examples are only examples of the present invention, and the present invention is not limited to the examples.

Example 1 Preparation of Blood Coagulation Factor VIII C2 Domain (FVIIIC2) Recombinant E. coli (Part 1)
Plasmid DNA was extracted / purified using Ultimate Miniprep Kit I (Omega BIO-TEK) from Ultimate ^™ Human ORF CLONE IOH10704 (Invitrogen), and using this as a template, primers F8C2F4 shown in SEQ ID NO: 3 and PCR was performed using the primer F8C2R2X shown in SEQ ID NO: 4 to amplify DNA containing the human FVIIIC2 gene. PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) was used for PCR. By treating the amplified DNA with SacI and XhoI, incorporating it into the cloning vector pET28a (Novagen) treated with SacI and XhoI, and transforming E. coli JM109 competent cells (Takara Bio) by the heat shock method Recombinant E. coli JM109 / pF8C2_11 was obtained.

Example 2 Nucleotide Sequence Analysis of FVIIIC2 Gene Recombinant Plasmid Plasmid pF8C2_11 was extracted from recombinant E. coli JM109 / pF8C2_11 obtained in Example 1 using Plasmid Miniprep Kit I (Omega BIO-TEK) and used as a template The base sequence was determined from the primer T7 terminator shown in SEQ ID NO: 5 by the dideoxynucleotide method, and as a result, the base sequence of SEQ ID NO: 1 was obtained.

Example 3 Preparation of FVIIIC2 transgenic E. coli (part 2)
Recombinant Escherichia coli BL21 (DE3) -RIPL is transformed by transforming competent cells of E. coli BL21-CodonPlus ^™ (DE3) -RIPL (Stratagene) with the heat shock method using the pF8C2_11 obtained in Example 1. / PF8C2_11 was obtained.

Example 4 Expression of FVIIIC2 protein from FVIIIC2 gene recombinant E. coli FVIIIC2 protein was expressed using recombinant E. coli BL21 (DE3) -RIPL / pF8C2_11 obtained in Example 3 according to the following method.
(1) Cell density is 37 ° C. at 37 ° C. in LB medium (40 mL) containing BL21 (DE3) -RIPL / pF8C2 — 11 and 20 μg / mL each of kanamycin and chloramphenicol. D. Shake culture was performed at ₆₀₀ to 1.0 to 1.2.
(2) IPTG was added to the culture of (1) so that it might become 1 mM, and FVIIIC2 protein was expressed by carrying out the shaking culture on the following conditions.

a) 5 hours at 25 ° C. b) 4 hours at 29 ° C. c) 3 hours at 33 ° C. d) 3 hours at 37 ° C. (3) After culturing under each condition, the culture is ice-cooled, and each is cooled at 4 ° C. Was centrifuged (10000 rpm, 10 minutes) to remove the culture solution.
(4) After repeated centrifugation to completely remove the culture solution, the bacterial pellet was suspended in 5 mL of Lysis buffer (composition: 20 mM potassium phosphate buffer (pH 7.0) / 200 mM NaCl / 40 mM imidazole). It became cloudy and the bacteria were crushed with an ultrasonic crusher under cooling at 4 ° C.
(5) Centrifugation (14000 rpm, 10 minutes) of the bacterial crushing solution was performed at 4 ° C. to separate the supernatant and the precipitate, and the precipitate was resuspended in 5 mL of ion-exchanged water.
(6) A portion of the supernatant and the precipitate suspension was heat-treated in the presence of a reducing agent, and then applied to SDS-polyacrylamide (15% acrylamide) gel electrophoresis (SDS-PAGE) in an equal amount for analysis.

  The result of SDS-PAGE is shown in FIG. When the culture temperature was 25 ° C., a 23 kDa band corresponding to the FVIIIC2 protein was not clearly observed in either the supernatant fraction (soluble protein) or the precipitated fraction (insoluble protein). When the culture temperature was 29 ° C., 32 ° C., or 37 ° C., a 23 kDa band was observed in each of the supernatant fraction and the precipitate fraction, but when the culture temperature was 37 ° C., the ratio of the precipitate fraction was high. When the culture temperature was 29 ° C. and 32 ° C., the ratio of the supernatant fraction was high. When expressed as an insoluble protein, a complicated operation such as solubilization is required. Therefore, in order to produce a protein simply and efficiently, it is preferably expressed as a soluble protein. Therefore, it can be said that the culture temperature for expressing FVIIIC2 protein from recombinant E. coli is preferably 26 ° C to 35 ° C. In particular, when cultured at 29 ° C. and 32 ° C., it can be said that culturing at 29 ° C. to 32 ° C. is more preferable because a large amount of soluble protein was expressed.

  Further, in this example, the culture time for expressing the FVIIIC2 protein is 3 to 4 hours, which is a short time compared to the conventional technique (cultured at 16 ° C. for 16 to 20 hours, Non-Patent Document 6). Degradation of the FVIIIC2 protein can also be suppressed.

Example 5 Purification of FVIIIC2 protein FVIIIC2 protein was purified using the recombinant E. coli BL21 (DE3) -RIPL / pF8C2_11 obtained in Example 3 according to the following method.
(1) Cell density is 37 ° C. at 37 ° C. in LB medium (2 L) containing BL21 (DE3) -RIPL / pF8C2 — 11 and 20 μg / mL each of kanamycin and chloramphenicol. D. Shake culture at ₆₀₀ until 1.2.
(2) IPTG was added to the culture of (1) so as to have a concentration of 1 mM, and FVIIIC2 protein was expressed by shaking culture at 30 ° C. for 3 hours.
(3) The culture was ice-cooled and centrifuged at 8000 ° C. (8000 rpm, 30 minutes) to remove the culture solution.
(4) The bacterial pellet was suspended in 30 mL of Lysis buffer, centrifuged at 4 ° C. under cooling (8000 rpm, 30 minutes) to remove the supernatant, and the bacterial pellet was resuspended in 120 mL of Lysis buffer. . This was crushed by an ultrasonic crusher while cooling at 4 ° C.
(5) The bacterial crushing solution was centrifuged (9000 rpm, 40 minutes) under cooling at 4 ° C. to obtain a supernatant.
(6) An equal volume of 4M urea was added to the supernatant, and the solvent composition was 10 mM potassium phosphate buffer (pH 7.0) / 100 mM NaCl / 20 mM imidazole / 2M urea.
(7) Adsorb / wash buffer solution (composition: 20 mM potassium phosphate buffer (pH 7.0) / 500 mM NaCl / 40 mM imidazole / 2 M urea) with 5 mL of HisTrap HP (manufactured by GE Healthcare) packed in a metal chelate chromatography packed column. Equilibrate and pass the whole amount of the bacterial extract prepared in (6), and then wash the column by passing 70 mL of adsorption / washing buffer to remove non-specific adsorbents. did.
(8) Prepare an elution buffer (composition: 20 mM potassium phosphate buffer (pH 7.0) / 500 mM NaCl / 400 mM imidazole / 2M urea), and establish a linear concentration gradient that makes the ratio of the elution buffer 0 to 100%. The eluted protein was monitored over time. For this operation, AKTAPlime, which is a medium-low pressure chromatography system manufactured by GE Healthcare, was used (FIG. 2).
(9) The peak fraction of the eluted protein was analyzed by SDS-PAGE (15% acrylamide) under reducing conditions (FIG. 3), and the highly purified part (the 36th to 41st fractions in FIG. 3) was collected. And dialyzed against a dialysis buffer (composition: 10 mM Tris-hydrochloric acid buffer (pH 7.5) / 1 mM EDTA, 100 mM NaCl) to obtain a final sample. The final sample was dispensed and stored at −80 ° C., and the concentration was assayed by the Bradford method using BSA as a standard protein (protein concentration: 0.7 mg / mL). The amino acid sequence of the final sample protein is shown in SEQ ID NO: 2.

Example 6 Confirmation of FVIIIC2 protein (1)
The protein preparation obtained in Example 5 was analyzed using SDS-PAGE (15% acrylamide) under non-reducing conditions. Two bands were detected when heat treatment was performed at 98 ° C. for 5 minutes before applying to the gel, and one band was detected when heat treatment was not performed (FIG. 4). In the former, the upper thin band coincided with the band position under reducing conditions. This result shows that the protein sample obtained in Example 5 has a disulfide bond between two cysteine residues (the 40th and 192nd cysteines of SEQ ID NO: 2), and this bond is partially treated by heat treatment. It can be interpreted as a reductive cleavage.

Example 7 Confirmation of FVIIIC2 protein (2)
The FVIIIC2 binding peptide having the structure shown in FIG. 5 was chemically synthesized with reference to Non-Patent Document 2 (commissioned to Hayashi Kasei Co., Ltd.). The usual solid phase peptide synthesis method was used for the synthesis, and the N-terminus was protected with an acetyl group, and the 7th and 14th cysteine residues from the N-terminus were disulfide bonded. The sample was identified by mass spectrometry (HP 1100 series LC / MSD: manufactured by Hewlett Packard), and the purity test was performed by HPLC (Shiseido capcell pak C18 column: manufactured by Shiseido Co., Ltd.) to confirm a purity of 99% or more. This peptide is dissolved in DMSO, immobilized on a CM5 sensor chip of BIACORE2000, which is an SPR analyzer, by the amine up-ring method, and the protein sample obtained in Example 5 is diluted to 3.0 μM with HBS-EP and analyzed. The sensorgram shown in FIG. 6 was obtained by analyzing as light. By analyzing this with the BIAEvaluation program, 1.0 × 10 ⁻⁷ M was obtained as a dissociation constant (KD) representing the interaction between the two. From these results, it was shown that the FVIIIC2 protein produced by the method of the present invention has an affinity equivalent to that of the natural FVIIIC2 protein.

SDS-PAGE analysis showing expression and localization of FVIIIC2 at each culture temperature. In the figure, S indicates the supernatant fraction (soluble protein), P indicates the precipitated fraction (insoluble protein), and the arrow indicates the band corresponding to the FVIIIC2 protein (23 kDa). Protein elution profile with imidazole concentration gradient. SDS-PAGE analysis of each fraction in FIG. The arrow in the figure indicates a band corresponding to FVIIIC2 protein (23 kDa). SDS-PAGE analysis of purified FVIIIC2 protein under non-reducing conditions. Structure of FVIIIC2 binding peptide. The interaction analysis result (BIACORE sensorgram) of FVIIIC2 binding peptide and purified FVIIIC2 standard.

Claims (4)

In the method for producing a blood coagulation factor VIII C2 domain protein using a recombinant Escherichia coli into which a polynucleotide containing a blood coagulation factor VIII C2 domain gene is introduced, the blood coagulation factor VIII C2 domain is incorporated into the cells of the recombinant E. coli. Urea is added within the range of 1M to 3M to the intracellular soluble fraction after the protein is expressed, and then the metal chelate chromatography is performed in the presence of urea within the range of 1M to 3M. A method for producing a blood coagulation factor VIII C2 domain protein, which comprises purification. The initial concentration of imidazole is in the range of 20 to 50 mM and the final concentration is 200 to 500 mM with respect to the soluble fraction in the cells after the blood coagulation factor VIII C2 domain protein is expressed in the cells of the recombinant E. coli. The method for producing a blood coagulation factor VIII C2 domain protein according to claim 1, wherein the protein is eluted and purified from a metal chelate chromatography carrier by applying a concentration gradient within the range of. The polynucleotide containing the blood coagulation factor VIII C2 domain gene is obtained by adding at least a histidine tag sequence to the 5 ′ end side of the blood coagulation factor VIII C2 domain gene. A method for producing a blood coagulation factor VIII C2 domain protein according to claim 1. The method for producing a blood coagulation factor VIII C2 domain protein according to claim 1 or 2, wherein the polynucleotide containing the blood coagulation factor VIII C2 domain gene is a polynucleotide consisting of SEQ ID NO: 1.

Images