JP2774154B2 - Activated human protein C derivative - Google Patents

Description

[Detailed Description of the Invention] [Object of the Invention] The field of industrial application The human protein C derivative or the activated human protein C derivative according to the present invention is used as an anticoagulant, for example, venous thrombosis, generalized intravascular coagulation ( DIC) and other diseases caused by thrombus formation. It is also intended to be used as a fibrinolysis accelerator as an adjuvant to fibrinolysis. The activated human protein C derivative or the human protein C derivative activated in the body of the present invention has a longer half-life in blood than natural type activated human protein C, and thus has a feature of maintaining its effect more.

2. Description of the Related Art A living body is provided with a mechanism for preventing bleeding, which is undesirable for itself, from occurring, that is, a blood coagulation system. This system is mainly composed of a group of plasma proteins, blood cells such as platelets, and vascular endothelial cells, and stops blood by forming a fibrin network containing blood cell components and the like. However, when this system does not need it,
Alternatively, when operated at a site that is not required, a thrombus that is inconvenient to the self may be generated, and sometimes fatal. On the other hand, plasma also has a system for controlling this blood coagulation system. One of the major ones is antithrombin III, an inhibitor of the blood clotting factor thrombin. Another is the protein C mediated system, which acts at an earlier stage of the blood coagulation system.
Protein C is a kind of plasma serine protease precursor, and is activated on the surface of platelets and vascular endothelial cells by limited degradation by a complex of thrombin and its receptor, thrombomodulin, and activates protein C, a serine protease. (APC). ¹⁾ APC
Exerts an anticoagulant activity by selectively degrading the activated factor 5 and the activated factor 8 of the blood coagulation system.
^{2), 3)} It is known that this activity is enhanced by protein S. ⁴⁾ APC is also thought to have a fibrinolytic promoting effect by cleaving PAI-1 which is an inhibitor of tissue plasminogen activator. ⁵⁾ Human protein C is biosynthesized in the liver and approximately 4μ
g / ml is present. It is known that human protein C undergoes various post translational modifications during its biosynthesis in its secretory cells. This includes removal of the pre-region (positions −42 to −25) and pro-region (positions −25 to −1), which are signal peptides, nine γ-carboxylations, and β-hydroxylation at the 71st position. Furthermore, it is secreted extracellularly after undergoing modifications such as N-glycosylation, which seems to be at four places, and double-chain formation by removing Lys at position 156 and Arg at position 157. [gamma] -Carboxylation is a process requiring Vitamin K, and the sequence of the pro region is related to this, and it has been revealed that the portion from position -12 to position -17 is particularly important. ⁶⁾ This γ-carboxyglutamic acid (Gla) has a binding property to calcium ions and is necessary for the expression of APC activity.

Human protein C has a light chain (molecular weight: about 21,000) composed of a Gla domain and an epidermal growth factor (EGF) -like domain, and a heavy chain (molecular weight: about 41,000) composed of an activation peptide and a catalytic domain. Disulfide-bonded (double-chain type), but a small amount of 1
There is also main chain protein C. Upon conversion to APC, the 12-residue activation peptide is removed and the conformation changes accordingly. APC exerts its anticoagulant activity not only on the catalytic domain but also on the light chain. The light chain is a region necessary for binding to a phospholipid membrane via calcium ions and for synergistic action with protein S.

On the other hand, APC is known to be inhibited by plasma inhibitors, and this is thought to be related to the half-life of APC activity in blood. The main ones are protein C inhibitor (PCI) ⁷⁾ and alpha 1 antitrypsin ⁸⁾ .

Human protein C or human APC appears to be effective in treating those deficient and may be used in the treatment of various diseases caused by thrombus formation. This includes, for example, venous thrombosis, generalized intravascular coagulation (DIC). It may also be used as an adjunct to fibrinolysis.

As a method for producing human protein C, there is a method of purifying from human crystals, and by activating it with an activator, human APC can be produced. However, production by recombinant DNA technology has been studied due to the problem of supply of human plasma as a raw material and concerns about possible contamination with AIDS virus, hepatitis virus and the like. Encodes human protein C. The cDNA has already been revealed. ^{9), 10)} Expression of protein C in animal cell hosts using this has already been performed. ^{11) In} addition, an expression method in animal cells in which the ratio of double-strand formation was increased by modifying the vicinity of the cleavage site for double-strand formation was also developed. ¹²⁾ Also disclosed is a method for direct expression of APC. ¹³⁾ Problems to be Solved by the Invention A problem when activated human protein C is used as a therapeutic agent is that its half-life in blood is as short as about 20 minutes. Therefore, there is a difficulty in administering this effectively by infusion.

As described above, the main factor that determines the half-life of activated human protein C in blood is protein C inhibitor (PC
I), it is thought to be in the interaction with inhibitors in plasma such as α ₁ -antitrypsin. Therefore, in order to obtain activated human protein C having a long half-life, it was considered that a derivative having reduced reactivity with such an inhibitor should be prepared. These inhibitors ultimately form a covalent bond with the active center serine residue, but may require access to the active center as a pre-step. The active center portion of serine proteases, including activated human protein C, is thought to lie deep within its "hollow" in the protein's conformation. Thus, it is likely that such inhibitors first interact with the "edge" of the "well". ^{14 In} addition, we investigated this interaction mainly as a result of electrostatic interaction.

On the other hand, this interaction is viewed from the side of activated human protein C which is a protease. Activated human protein C contains regions necessary for the expression of activity as a serine protease (such as Asp-His-Cer residues in the active center and amino acid residues that fix them in an appropriate spatial position), There may be regions involved in the recognition of specific substrates. In addition, it is likely that there are regions involved in the interaction with specific inhibitors. However, it is not clear whether this region is different from the region involved in substrate recognition, but we considered that this region was a part corresponding to the "edge" of the aforementioned "dent". From the above viewpoint, a human protein C derivative as shown in FIG. 1 was prepared by a protein engineering technique. The modification sites in Nos. 4, 5, and 6 are “consensus” amino acid acid groups that are commonly found in the corresponding parts of other serine proteases, and are therefore necessary for the expression of the aforementioned serine protease activity. Was considered to be equivalent to an area. On the other hand, the other three modification sites are amino acid residues unique to activated human protein C, and thus were considered to be parts that may be involved in the interaction with the inhibitor. .

Means for Solving the Problems The present invention provides the following three conditions in the amino acid sequence of activated human protein C: (i) in the three-dimensional structure of activated human protein C protein, the active center His (42), Asp (88) and Ser
An amino acid residue corresponding to the “edge” of the cavity where (191) is located (however, the number is 1 at the N-terminus of the heavy chain); (ii) a “consensus” amino acid residue of serine proteases And (iii) an activated human protein C derivative having a sequence in which one or more of the amino acid residues satisfying the charged amino acid residue is replaced with an oppositely charged essential amino acid residue; and This was made by studying a human protein C derivative in which an activation peptide is bound to the N-terminal of the heavy chain of the activated human protein C derivative. Specifically, in a protein having the structure of native activated human protein C, an activated human protein C derivative having a structure in which any one of the following substitutions is made; The activated human protein C derivative is a human protein C derivative.

A. Substitution with the Asp residue at the 45th residue from the amino terminus of the heavy chain as an Arg residue B. Substitution with the Asp residue at the 182th residue from the amino terminus of the heavy chain as an Arg residue C. Heavy chain Substitution of the Arg residue at position 183 from the amino terminus with an Asp residue The present invention also provides an activator by treating the above human protein C derivative with an enzyme such as thrombin, thrombin-thrombomodulin complex or snake venom. The present invention provides a method for obtaining the activated human protein C derivative by excision of an isotropic peptide.

The production of the activated human protein C derivative shown in the present invention is performed using recombinant DNA known as protein engineering.
Achieved by technology. One of the methods for modifying the cDNA of human protein C uses site-directed mutagenesis, which is specifically described in Examples. Alternatively, the cDNA can be modified using a method known as "cassette mutation method". This is a method in which a cDNA fragment containing a portion to be modified is removed using an appropriate restriction enzyme, and a DNA fragment containing a sequence to be modified produced by a chemical synthesis method is inserted instead.

Various methods for expressing a desired cDNA in a host cell are known. Basically, a DNA sequence related to transcription called a promoter, which has an action in the host cell,
It is achieved by ligating with the cDNA of interest and introducing it into the host cell in some way. A host cell for carrying out the present invention must be selected from at least eukaryotic cells in the sense that it is expected that modifications such as removal of a propeptide, addition of a sugar chain, and α-carboxylation are correctly performed.

In the examples, there is known a method for obtaining an activated human protein C derivative by expressing a human protein C derivative and then activating it with a protein C activator derived from snake venom. Alternatively, thrombin or thrombin-thrombomodulin complex can be used as the activator. Alternatively, by simultaneously modifying the activation peptide region, it is possible to express the protein in the form of an activated human protein C derivative from the beginning.

EL for human protein C or activated human protein C
Quantitation by ISA has already been developed. ¹⁵⁾ The human protein C derivative of the present invention or activated human protein C
This can be applied to the quantification of derivatives. In this method, only human protein C derivatives having Gla or activated human protein C derivatives can be measured.

For the production of the human protein C derivative or activated human protein C sugar of the present invention, purification methods of natural human protein C including barium adsorption method and ion exchange chromatography method can be applied. It is particularly preferred to use affinity column chromatography using an anti-human protein C monoclonal antibody that recognizes the conformational change of the Gla domain due to. ¹⁶⁾ is an excellent method in that only those having Gla and thus having anticoagulant activity can be purified, and a mild eluent called EDTA can be used.

Example In the example of the present invention, the following method was used.

Cleavage of DNA 1 μg of plasmid DNA or replicative form (RF) DNA or DNA fragment of M13 phage can be digested with 10 μg.
This was performed by using 4 to 10 units of restriction enzyme in μ buffer and keeping at the temperature specified by the manufacturer for 2 hours. The buffer used was the one attached to the restriction enzyme.

Recovery of DNA Fragment from Agarose Gel DNA fragments cut with restriction enzymes were separated by 0.8% agarose gel electrophoresis using a submarine type electrophoresis tank. Cut out agarose gel containing the target DNA fragment,
Collected using ENECLEAN (Bio 101). The method followed the attached instructions.

DNA fragment ligation was performed using a DNA ligation kit (Takara Shuzo). The method followed the attached instructions.

E. coli transformation E. coli HB101 competent cells (Takara Shuzo)
A DNA solution of μ or less was added and kept on ice for 1 hour. Then 4
After soaking in a 2 ° C. water bath for 1 minute, it was placed again on ice for 5 minutes. This was added to 1 ml of L-broth, and cultured with shaking for 1 hour. A part (50 μ-300 μ) of the mixture was placed on an ampicillin plate (L-broth, agar 15 g /, ampicillin 50 μm).
g / ml) and cultured overnight at 37 ° C to form colonies.

Small-scale preparation of plasmid DNA Preparation was performed by the alkaline lysis method. Specifically, “Molecular Cloning” (T. Maniatis, Cold Spring Harbo
r Laboratory, 1982), p. 368. Purification by "GENECLEAN" described above was performed as necessary.

Large-scale preparation of plasmid DNA Basically, "Transcription and Translation" (BD
Hames, IRL press, 1984), alkaline lysis and CsCl equilibrium density gradient centrifugation according to the procedure described on page 8. However, the rotor used for ultracentrifugation was Hitachi RP-67VF
It is a vertical rotor. The removal of CsCl was not replaced by dialysis, but by diluting 4-fold with TE (10 mM Tris · HCl pH 8.0, 1 mM EDTA), followed by ethanol precipitation.

Determination of DNA base sequence dideoxy chain termination (dideox
y chain termination) method. The reagent used in the reaction was that of Takara Shuzo's "7-deaza sequence kit", and the operating procedure was in accordance with the instructions attached to the kit. ³⁵ S-CTPαS (400 Ci / mmol, Amersham) was used for the label. Electrophoresis is 0.3m of 6% acrylamide
Using a gel having a thickness of m, the gel was dried after electrophoresis, and then subjected to autoradiography. The temperate DNA used was used since single-stranded DNA was easily obtained after the modification of human protein C cDNA. In other cases, the double-stranded plasmid was neutralized with alkali after denaturation. Using. The procedure at that time is described in "Vector DNA" (Yoshiyuki Sakaki, Kodansha, 1986)
According to the method described on page 67. As the primer, 18 bases of the sequence in the vicinity of the part to be examined were chemically synthesized, and purified and used.

Chemical synthesis of DNA fragment and its purification Primers for cDNA modification (Fig. 4) and primers for deferred sequencing were synthesized on an Applied Biosystems 380A DNA synthesizer under the conditions of "Tr ON, AUTO". did. For the purification, an “oligonucleotide purification cartridge” manufactured by the company was used according to the attached instructions.

Example 1 Obtaining cDNA Encoding Human Protein C mRNA was extracted from human hepatocytes according to the guanidine thiocyanate method ¹⁷⁾ . 5 x GT for 2 x 10 ⁸ human hepatocytes
C solution (6 M guanidinium isothiocyanate, 5 mM sodium citrate, 0.1 M2-mercaptoethanol, 0.5%
Sodium N-lauroyl sarcosinate) was added and homogenized. This was overlaid on 3.8 ml of 5.7 M CsCl, 0.1 M EDTA aqueous solution, and ultracentrifuged at 25 ° C. for 15 hours at 35,000 rpm using an RPS-40T rotor (manufactured by Hitachi). After ultracentrifugation, the solution was carefully removed, rinsed three times with about 1 ml of ethanol, dissolved in 1.4 ml of water and precipitated with ethanol.
This precipitate was washed with 0.5 M NaCl, 10 mM Tris · HCl (pH 7.5), 1 mM
After washing with the composition of EDTA and 0.05% SDS, dissolve in 0.5 ml and add 0.5 ml
Oligo (dT) cellulose column.

After washing the column with the above washing solution, 10 mM Tris-HCl
(PH 7.5), elution was performed with an eluate having a composition of 1 mM EDTA and 0.05% SDS, to obtain about 31 μg of polyA ⁺ RNA. Based on this,
According to r and Hoffman's method ¹⁶⁾ , cDNA was synthesized using a cDNA synthesis kit manufactured by Amersham.

5 units of polyA ⁺ RNA and 50 units of human placenta-derived RNase
In the presence of the inhibitor (HPRI), 5 μg of Oligo (dT) 12-18 was added, and 100 units of reverse transcriptase were allowed to work at 42 ° C. for 1.5 hours to synthesize a single-stranded cDNA at a yield of about 30%. 4
Units of Escherichia coli ribonuclease H and 115 units of Escherichia coli DNA polymerase I were added, reacted at 12 ° C for 1 hour and at 22 ° C for 1 hour, and left at 70 ° C for 10 minutes to inactivate the enzyme. Then add 10 units of T4 DNA polymerase 37
The mixture was reacted at 10 ° C. for 10 minutes to obtain a double-stranded cDNA at a yield of about 95%. 20 units of EcoRI methylase were added to this double-stranded cDNA.
After acting at 37 ° C. for 1 hour, EcoRI linker (Takara Shuzo) was bound. After adding 16 units of EcoRI (Takara Shuzo) and reacting at 37 ° C for 2 hours, Sepharose CL
Through a −4B column, about 0.8 μg of purified cDNA was obtained.
Next, in vitro packaging was carried out using 0.4 μg of this cDNA and 1.0 μg of λgt10 arm (manufactured by Vector Cloning Systems), and human hepatocyte-derived cD
An NA library was obtained. E. coli C600
The hfl ^- strain was infected and formed plaques. Clones containing the human protein C gene were selected by the plaque hybridization method using the following ³² P-labeled synthetic DNAs PC-1 and PC-2 as probes.

From λgt10 phage containing cDNA for human protein C
DNA was prepared according to the method of Thomas and Davis. This DNA was digested with EcoRI and subjected to agarose gel electrophoresis to recover a cDNA fragment of human protein C. This DNA
PUC8-PC1 was constructed by ligating the fragment with PUC8 previously treated with EcoRI and treated with bacterial alkaline phosphatase. When the nucleotide sequence of this human protein C cDNA was examined, it was found that the 5 ′ portion of the human protein C cDNA lacked about 50 base pairs as compared with the full-length human protein C cDNA. Supplemented.

Example 2 Construction of Native Human Protein C Expression Vector As shown in FIG. 2, PUC8-
Hind III and Sac, each with one cleavage site on PC1
Cleavage with Sac II
-A small fragment of Hind III was recovered. (This is referred to as A fragment.) PUC8-PC1 was digested with SacII and EcoRI and subjected to agarose gel electrophoresis.
-The Sac II fragment was recovered. This DNA fragment was further cut with Dde I having one cleavage site in this fragment, and then subjected to agarose gel electrophoresis to recover a Dde I-Sac II fragment. (This is referred to as a B fragment.) On the other hand, the DNA fragment from the upstream of the translation initiation site to the above-mentioned Dde I was cut with Hind III on the 5 ′ side and Dde on the 3 ′ side.
It was synthesized by the chemical synthesis method in the form cleaved with I. (This is referred to as C fragment.) Next, one Hind III and one Sa
pNAK having a cII cleavage site is cut with HindIII and SacII, and subjected to agarose gel electrophoresis to obtain HindI.
The II-Sac II fragment (containing the ampicillin resistance gene and origin of replication in E. coli) was recovered. This DNA fragment was ligated with the above-mentioned B and C fragments in three molecules to construct pNAK-PCU. Next, this pNAK-PCU was converted to Hind I
The fragments were cleaved with II and Sac II and subjected to agarose gel electrophoresis to recover a Hind III-Sac II small fragment. On the other hand, pSV2-gpt ¹⁹⁾ was partially digested with Apa I and subjected to agarose gel electrophoresis. One of the two Apa I sites was obtained.
The product which had been cleaved at only one site (linear type) was recovered, further subjected to complete digestion with Hind III, and then subjected to agarose gel electrophoresis again to recover the Hind III- (Apa I) -Apa I fragment shown in FIG. The DNA polymerase I Klenow fragment and deoxyribonucleotide triphosphate were allowed to act on this fragment,
Hind III site and Apa I site were blunt-ended.
After ligation of a phosphorylated Hind III linker (Takara Shuzo) to this DNA fragment, Hind III treatment was performed,
The sample was recovered again by agarose gel electrophoresis. This DNA
Escherichia coli HB101
To increase this. The plasmid DNA extracted from Escherichia coli was again cut with HindIII, treated with bacterial alkaline phosphatase, and ligated with the above-mentioned A and D fragments in three molecules to obtain a natural human protein C expression vector pSV2-PC1. .

Example 3 Construction of Human Protein C Derivative Expression Vector As shown in FIG. 3, pSV2-PC1 constructed in Example 2 was replaced with H
The fragment was cut with ind III and subjected to agarose gel electrophoresis to recover a human protein C cDNA portion. This DNA fragment is
Ligation was performed with M13 mp11 replicative form (RF) DNA previously treated with Hind III and bacterial alkaline phosphatase, and this was ligated to E. coli TG-
Introduced into one strain to generate plaque. The recombinant M13 phage was picked from this plaque and added to 2 ml of 2 × TY medium together with 20 μg of an overnight culture of E. coli TG-1 strain, followed by shaking culture at 37 ° C. for 5 hours. Escherichia coli in this culture solution is collected, replicative form (RF) DNA is prepared by the alkaline lysis method, and analyzed by restriction enzyme digestion.
A clone in which the human protein C cDNA was inserted in the opposite direction to the M13 gene was identified. From recombinant M13 phage in the culture supernatant when this clone was cultured, Amersham “Oligonucleotide directed in vitro mutagenesis”
The template DNA was prepared according to the procedure attached to the “system.” Using the template DNA and the primers shown in FIG. 4, “Aligonucleotide directed by Amersham, Inc.
Using an in vitro mutagenesis system according to the instructions attached thereto, a human protein C derivative
cDNA was prepared. This is basically based on the method of F. Eckstein et al. ²⁰⁾ M13 phage replicative form DNA containing the obtained human protein C derivative cDNA
Was introduced into E. coli TG-1 to generate plaques. 2 ml of the recombinant M13 phage in this plaque was
The recombinant M13 phage was prepared from the culture supernatant by polyethylene glycol precipitation, and treated with phenol to obtain single-stranded DNA. Specifically, Amersham “M13 cloning and sequen
I followed the handbook attached to the "cing kit".
The base sequence was determined using NA as a template and the 18 bases (+ strand) in the vicinity of the chemically modified region as primers, and clones having the desired modification were selected. From the cells corresponding to the clone that has been preserved, the replicative M13 phage
Foam (RF) DNA was prepared. This is Bio101 “GENEC
LEAN "and purified with Sac II and Nae I (No. 1) or Ava I
(Nos. 2 to 7) were digested and subjected to agarose gel electrophoresis to recover a DNA fragment containing a modified portion. Also pSV2
-PC1 is also Sac II and Nae I (No. 1) or Ava I (No.
Digested in 7) and treated with bacterial alkaline phosphatase were subjected to agarose gel electrophoresis, and the larger fragment was recovered. The DNA fragment containing these modified portions and the pSV2-PC1 DNA fragment cut with the same restriction enzymes were ligated and introduced into Escherichia coli HB101 strain. Plasmids were prepared from the obtained transformants by the alkaline lysis method, cut with Hind III and the restriction enzymes used for recombination, and analyzed by agarose gel electrophoresis to select those having the desired recombination. In addition, these plasmids
The nucleotide sequence in the vicinity of the modified portion was examined to confirm that the correct recombination was performed. In addition, M13 DNA was used for the entire region of the DNA fragment containing the modified portion used in this recombination.
The nucleotide sequence was examined in the state of being integrated into the plasmid or in the form of a plasmid in the form of an expression vector, and it was confirmed that unintended modification did not occur. A plasmid was extracted from Escherichia coli containing the thus obtained expression vector and cultured in 400 ml of L-broth, and used for expression in Example 4 as a human protein C derivative expression vector.

Example 4 Expression of Human Protein C Derivative Cos-7 cells (ATCC CRL-1651) were cultured using a Falcon 3025 dish in 30 ml of 10% FCS-eRDF per dish. Twelve cells were detached from a nearly confluent petri dish by trypsin treatment, and the suspension in 60 ml of PBS (-) was centrifuged at 1,000 rpm for 5 minutes at room temperature to remove the supernatant. The suspension was again suspended in 60 ml of PBS (-), centrifuged at 1000 rpm for 5 minutes at room temperature, and the supernatant was removed to obtain 0.9 ml of PBS (-).
Suspended in water. On the other hand, a human protein C derivative expression vector
96 μg was previously sterilized by ethanol precipitation in a 1.5 ml Eppendorf tube, and
pm After centrifugation for 10 minutes, aseptically remove the supernatant, and add PBS (-) 0.6 ml
Was dissolved. This was mixed with the above-mentioned suspension of Cos-7 cells (to approximately 2.4 ml), and “GENE PULSE” manufactured by Bio-Rad.
R "cuvettes (0.8 ml) were dispensed into three tubes. Electroporation was performed using Biorad" GENE PULSER "under the conditions of 1000 V and 25 μF once. The cell suspension was immediately transferred to a Falcon 2059 tube. Transfer, 10 ml of 10% FCS
Gently shaken dropwise -eRDF-2μg / ml vitamin K _1, and diluted. 30 ml of 10% FCS-eRDF-2 μg
₁ in 12 Falcon 3025 Petri dishes containing ₁ / ml Vitamin K
Each ml was added and diluted. This in a CO ₂ incubator 2
After 4 hours incubation, the petri dish per 5 ml PBS (-) Wash 2 times with, ITES-eRDF-2μg / ml serum-free medium of vitamin K ₁ added petri dish per 30 ml, and further cultured for 48 hours, the culture supernatant Collected. (ITES: 9 μg / ml insulin, 10 μg / ml
Transferrin, 10 μM ethanolamine 2 × 10 ^-8 M
Selenite, eRDF: Far Seiyaku) the ITES-eRDF-2μg / ml vitamin K ₁ of the same amount was added, again
The cells were cultured for 48 hours, and the operation of collecting the culture supernatant was performed twice more. Thus, a culture supernatant containing the modified human protein C was obtained.

The concentration of the human protein C derivative in the culture supernatant or the concentration of the human protein C derivative having Gla was measured by a side-switch ELISA method. JTC-4 (H chain recognition) was used as the monoclonal antibody on the plate side, and JTC-4 was used as a horseradish peroxidase (HRPO) -labeled antibody.
-1 (for recognizing the Gla domain depending on Ca ²⁺ and for measuring human protein C derivatives having Gla) or JTC-5 (for recognizing activated peptides and for measuring the entire human protein C derivative) was used. ^{21) At the time of} measurement, a calibration curve was drawn using human protein C derived from plasma of American Diagnostica. Table 1 shows the concentration of the human protein C derivative in the culture supernatant.

Example 5 Purification of human protein C derivative The culture supernatant obtained in Example 4 was passed through a membrane filter (Millipore) to remove cell debris, and then 5 mM C
aCl ₂ , 0.02% NaN ₃ 25 units / ml aprotinin and 1 mM benzamidine were added (all concentrations were final concentrations). A column of ~ 1 ml bed volume was prepared by coupling JTC-3 ²¹⁾ to Cellulofine in advance, and 5 mM CaCl ₂ -TB
It had been equilibrated with S (pH7.4) -0.02NaN _3. 20ml for this
/ h, add the culture supernatant at 4 ° C, and add 5mM under the same conditions.
The plate was washed with CaCl ₂ -1M NaCl-50mM Tris · HCl (pH 7.4) for 4 hours. Elution was performed with 8 ml of 10 mM EDTA-TBS (pH 7.4) -0.02% Na
It was performed at 20ml / h in N _3. Next, CaCl ₂ was added in an amount of 10 mM (final) larger than the amount corresponding to EDTA, and further concentrated to about 300 μm with Centricon 10 (Amicon). An aliquot was diluted and its concentration measured by ELISA.

Example 6 Synthetic substrate cleavage activity of activated human protein C derivative 0.5 purified human protein C derivative obtained in Example 5
To μg, 100 μl of 3 mM CaCl ₂ -TBS (pH 7.4), 10 μg of thrombin and 24 ng of thrombomodulin were added, and
Activated human protein C derivative was obtained by keeping at 1 ° C. for 1 hour. This was serially diluted with 3 mM CaCl ₂ -TBS (pH 7.4), and 2 ml of 0.1 M CsCl-50 mM Tris.HCl (pH 8.0) and 20 μm of 10 mM Boc-Leu-Ser-Thr-Arg-MCA ( Activated protein C activity measurement synthetic substrate)
, And the fluorescence at 440 nm was measured to examine the activity of these activated human protein C derivatives to cleave the synthetic substrate. The results are shown in FIG. Derivatives of Nos. 4, 5, and 6 had significantly reduced specific activities, whereas derivatives of Nos. 1, 2, and 3 had specific activities that were not so different from the natural type.

Example 7 Anticoagulant Activity of Activated Human Protein C Derivative (Arg (183) → Asp) Derivative of Human Protein C Purified in Example 5 (Arg
(183) → Asp) 2 μg of 160% with 0.1% BSA-TBS (pH 7.4)
, diluted with 40 µl of IU / ml Protac (American Diagnostica), and kept at 37 ° C for 1.5 hours.
Activated human protein C derivative. This was diluted and 12.5, 25, 50, 100, 200 ng / 50 μ0.1% BSA-TBS (pH 7.
4) This sample and 50 μm of Sysmex APTT reagent were added to 100 μm of Sysmex Control Plasma I kept at 37 ° C. for 2 minutes and stirred, and kept at 37 ° C. for 2 minutes, and 100 μl of 25 mM CaCl ₂ was added and stirred. APTT was measured using a Sysmex CA-100 blood coagulation analyzer. FIG. 6 shows the results of comparison with native activated human protein C. The derivative of Arg (183) → Asp showed about 60% specific activity of the natural type.

Example 8 Inactivation Rate of Activated Human Protein C Derivative in Plasma Plasma of three derivatives of Nos. 1, 2, and 3 whose synthetic substrate cleavage activity was not significantly different from that of the natural type in Example 6. The quenching rate in the medium was examined. 1 μg of the protein C derivative activated in the same manner as in Example 6 was added to 550 μm of human plasma, and 25 U / ml of aprotinin and 100 μM of MD805 (both were used)
(final) In the presence of 37 ° C. for 0, 5, 10, 20, and 60 minutes, 100 μ sampling was performed at each time, and the synthetic substrate cleavage activity was examined in the same manner as in Example 6. FIG. 7 shows data when the time when the time is 0 is set to 100. It can be seen that all the derivatives of Nos. 1, 2, and 3 have reduced inactivation rates.

Example 9 Inhibition Rate of Activated Human Protein C Derivatives by PCI Purified Protein C Inhibitor (PC
The rate inhibited by I) was compared to native APC. Human protein C or a derivative thereof was activated in the same manner as in Example 6, and the reaction was stopped by adding 100 μM (final concentration) of MD-805. 1 μg of purified PCI was added to each concentration of APC, 100
0.1% BSA-3mM CaCl ₂ -TBS (pH 7.5), 10μ
5 μg / ml dextran sulfate was added and reacted at room temperature for 30 minutes. The remaining APC activity was examined in the same manner as in Example 6, using the activity of cleaving the synthetic substrate as an index. The result is shown in FIG.
The horizontal axis shows the ratio of PCI / APC, and the vertical axis shows the residual APC activity. It can be seen that the derivative of No. 3 is less likely to be inhibited by PCI than the native APC.

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of native human protein C and the modified site studied in the present invention. FIG. 2 shows a construction process of a native human protein C expression vector. FIG. 3 shows a construction process of a human protein C derivative expression vector. FIG. 4 shows the nucleotide sequence of a synthetic primer used for modification of human protein C cDNA. FIG. 5 shows the synthetic substrate cleavage activity of the activated human protein C derivative. FIG. 6 shows an activated human protein C derivative (Arg (183) →
2 shows the anticoagulant activity of Asp). FIG. 7 shows the inactivation rate of activated human protein C derivative in plasma. FIG. 8 shows activated human protein C derivative (Arg (183) →
4 shows the inhibition rate of Asp) by a purified protein C inhibitor. References 1) Esmon CT, Proc Natl Acad Sci USA, 78, 2249 (198
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──────────────────────────────────────────────────続 き Continuing from the front page (72) Yataro Ichikawa 4-2-2 Asahigaoka, Hino-shi, Tokyo Teijin Limited Tokyo Research Center (72) Koji Suzuki 3- 18-15 Kurahonmachi, Tokushima-city, Tokushima Prefecture Inside the Tokushima University Enzyme Science Research Center (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 9/64 C12N 15/57 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] An activated human protein C having a structure in which any one of the following substitutions is made in a protein having the structure of a native activated human protein C:
Derivatives. A. Substitution with the Asp residue at the 45th residue from the amino terminus of the heavy chain as an Arg residue B. Substitution with the Asp residue at the 182th residue from the amino terminus of the heavy chain as an Arg residue C. Heavy chain Of the 183rd Arg residue from the amino terminus to Asp residue 2. A human protein C derivative which is enzymatically activated to become the activated human protein C derivative according to claim 1. 3. A method for obtaining an activated human protein C derivative by enzymatically activating the human protein C derivative according to the above item. A DNA encoding the activated human protein C derivative or the human protein C derivative according to claim 1 or 2. An expression vector for an activated human protein C derivative or a human protein C derivative having the DNA sequence according to claim 3. 6. A cell transfected with the expression vector according to claim 4.