JPH04210700A - Recombinant human thrombomodulin derivative - Google Patents

Abstract

NEW MATERIAL:Recombinant human thrombomodulin derivatives in which a serine-glycine-serine-glycine-glutamic acid fragment from the 472th amino acid to the 476th amino acid of human thrombomodulin and a continuous amino acid sequence in the neighborhood thereof are modified by removal, addition or substitution of amino acids not so as to be modified by a sulfonated glucosanoglycan breakable by chondroitinase A, B or C. USE:An anticoagulant. PREPARATION:For example, human chromosome DNA is partly digested by a restriction enzyme to produce a human gene library and human thrombomodulin gene is screened by carrying out hybridization thereof using a probe. From the obtained positive clone, DNA is separated and treated with a restriction enzyme. The restriction enzyme-treated DNA is then bonded to a vector and introduced into a host. Transformation is carried out and the resultant transformant is cultured, thus obtaining the objective recombinant human thrombomodulin derivative.

Description

[Detailed description of the invention]

[00013

[Industrial Field of Application] The present invention relates to recombinant human thrombomodulin derivatives. More specifically, recombinant human thrombomodulin derivatives with extended blood half-life by changing the amino acid sequence so as not to be modified by glycosaminoglycans that are cleaved by chondroitinase ABC, expression vectors thereof, and transformed cells thereof. It is related to. [0002] [0002] Thrombomodulin is a glycoprotein present on the membrane of vascular endothelial cells, and binds to thrombin to activate thrombin's fibrin coagulation activity, factor II and factor VIII, or It shows an inhibitory effect on platelet activation and also promotes the activation of protein C by thrombin (Experimental Medicine 6. (14), 139
6-1398 (1988); Pr. g, Hemost, Thromb,, 9.29-55
(1989) These physiological activities of thrombomodulin indicate that thrombomodulin is useful as an anticoagulant. The cDNA of human thrombomodulin has already been reported (EMBOJ, 6.1891-189).
7 (1987): Biochemistry, 26
．． 4350-4357 (1987)), and it has also been found that the human thrombomodulin gene does not have introns in the DNA of the gene on the chromosome (Proc, Natl, Acad, Sci, USA,
84.6425-6429 (1987): J, Bi
ochem, 103, 281-285 (1988)
), 5uzuki et al. 0, Biol, Chem,, 2
64.4872-4876 (1989)) produced this human thrombomodulin in cultured animal cells by genetic recombination, but no detailed study has been conducted regarding its structure. [0003] According to the revealed DNA sequence, human thrombomodulin is estimated to be composed of 557 amino acids. Human thrombomodulin functionally consists of 5 amino terminal regions, an EGF-like structural region, an O-glycosylation site region, a transmembrane region, and an intracytoplasmic region.
It has been reported that the EGF-like structure region is a region that acts on thrombin and promotes its ability to activate protein C (J, Biol, C.
hem,, 264, 4872-4876 (198
9) ). In addition, the amino terminal region, EGF-like structure region, and O-glycosylation site region are parts that exist outside the cell membrane, and human thrombomodulin, which is composed only of these parts, cannot bind to the cell membrane and is present in a solubilized form. Existing 0, Biol, Chem, 264.48
72-4876 (1989); Blood, Mutual;
1396-1399 (1990)). [0004] The present inventors focused on these three regions (amino terminal region, EGF-like structure region, and O-glycosylation site region) and attempted to produce recombinant human thrombomodulin consisting only of these three regions. . When the human thrombomodulin gene was modified, an expression vector was created, and an expression cell line was cultured by introducing this vector into cultured animal cells (CHO-Kl cells), a group containing sulfated glycosaminoglycans was detected in the culture medium. We were able to find a modified human thrombomodulin (Japanese Patent Application No. 1-269194.
International Application PCT/J P2O, 101342). This sulfated glycosaminoglycan structure is chondroitin-4-
It has sulfuric acid as its main structure, and has a structure that has not been previously known in human trobomodulin. However, when the blood half-life of recombinant human thrombomodulin containing this sulfated glycosaminoglycan was investigated in rats, it was found to be as short as about 20 minutes. [0005]

Problems to be Solved by the Invention When pharmaceuticals are used to treat diseases, it is extremely important that the blood concentration of the administered pharmaceuticals is maintained at an effective concentration over a long period of time. 1
In other words, in order to exert the expected medicinal effects, pharmaceuticals (
The blood half-life must be long enough. The same holds true for recombinant drugs, and various ideas have been made to prolong the half-life in the blood. [0006] The inventors have discovered the power of sulfated glycosaminoglycans.
The presence of sulfated glycosaminoglycans is responsible for the short half-life of recombinant human thrombomodulin in the blood.
When this recombinant human thrombomodulin was treated with chondroylanase ABC, the blood plasma time in rats was extended to 7.7 hours. This indicates that recombinant human thrombomodulin with a long blood half-life can be obtained by removing the sulfated glucosinoglycans attached to recombinant human thrombomodulin. [0007] Therefore, the inventors have discovered h
The site of sulfated glycosaminoglycan addition in recombinant human thrombomodulin was investigated. That is, after reducing recombinant human thrombomodulin, free SH groups were carboxyamidomethylated, this was completely digested with trypsin, and then Dowe
Ion exchange chromatography using xIX2 and alcohol precipitation were performed to separate peptide fragments containing sulfated glycosaminoglycans. When the N-terminal amino acid sequence of the obtained peptide fragment was examined, the addition site of the sulfated glycosaminoglycan was Val-Asp-Gly.
-Gly-Asp-3e r-Gly-X-Gly-G
lu-Pro-Pro-Pro (Val: valine, Asp: aspartic acid, GIY nigericin, Se
r: serine, Glu: glutamic acid, Pronibroline, X: not specified). This sequence matched the sequence starting from the 467th valine of human thrombomodulin. [0008] On the other hand, regarding the amino acid sequence around the binding site of glycosaminoglycan to the peptide chain, Set”
-Gly-Xaa-Gly or Gly-5et”
It has been proposed that the presence of -Gly and acidic amino acids in its vicinity is important (Xaa: any amino acid, Se
t” serine to which niglucosaminoglycan is attached)
(Proc, Natl, Acad, Sci, USA,
84. 3194-3198 (1987) + J,
Ce1l Biol, 1547-1556 (
(1989)), and although it is not known for human thrombomodulin, a part of rabbit thrombomodulin is modified with chondroitin sulfate-like/dermatan sulfate-like glycosaminoglycans, and it can be used to bind antithrombin III through the glycosaminoglycan moiety. It is estimated that the sulfated glycosaminoglycan exhibits a dependent antithrombin activity and that the binding site of the sulfated glycosaminoglycan is the serine-glycine-serine-glycine sequence in the O-glycosylation site region (J, Biol, Chem, 263.8
044-8052 (1988); Thr omb,
Res., 53.27-39 (1989)). [0009] Based on these findings, the present inventors determined that the sulfated glycosaminoglycan addition site of recombinant human thrombomodulin was 5er-Gly- from position 472 to position 476.
We predicted that this region would contain 8er-Gly-GIu, and by modifying the amino acid sequence of this region, we were able to create a recombinant human thrombomodulin derivative that was not modified with sulfated glycosaminoglycan. In addition, its blood half-life was extended to 7 hours in rats. The present invention was made based on this knowledge, and aims to provide a recombinant human thrombomodulin with a novel structure that has a long blood half-life and is useful as an anticoagulant. [00101

[Means for Achieving the Object] The purpose of the present invention is to solve the problem of a site predicted to be an addition site of sulfated glycosaminoglycan (serine/glycine from position 472 to 476 in the 0-glycosylation site region).・Remove the amino acid sequence (around serine, glycine, glutamic acid)
This is achieved using recombinant human thrombomodulin derivatives modified by additions or substitutions. [00111 This recombinant human thrombomodulin derivative was produced by site-directed mutagenesis of the DNA of the human thrombomodulin gene, including the serine, glycine, serine, glycine, and glutamic acid moieties from amino acid positions 472 to 476, and the consecutive amino acids surrounding it. A human thrombomodulin derivative gene modified by removing, adding, or substituting the DNA sequence encoding the sequence can be created and used for production. [0012] The human thrombomodulin gene is described in the literature (J, Biochem, 103.281
285 (1988)) is obtained from a human chromosomal gene using a probe that can be created based on the DNA sequence described in 285 (1988). This gene may be full-length, or may be a partial-length DNA encoding peptides in three regions: the amine terminal region, the EGF-like structure region, and the O-glycosylation site region, as described in the Examples. In other words, the minimum portion that does not impair the physiological activity of human thrombomodulin and has a site modified with sulfated glycosaminoglycan (probably the EGF-like structure region and the 472nd to 476th serine in the O-glycosylation region)・The length of the DNA may be within the range that encodes the amino acid sequence surrounding the glycine, serine, glycine, and glutamic acid parts. The gene for the human thrombomodulin derivative obtained was tagged with an appropriate promoter, terminator,
A signal sequence, etc., and, if necessary, an appropriate marker gene are added and then integrated into an appropriate vector. Vectors may be of any type as long as they function in cultured animal cells. For example, SV40 vector, R3Y (
Rous sarcoma virus) vector, MMTV (murine breast cancer virus) vector, or CMV (cytomegalovirus) vector. In addition, the cells used as host culture cells are not particularly limited, but include CHO
Cells and CO3 cells are particularly suitable. [Obtaining the human thrombomodulin gene of 0013l or more, constructing an expression vector, and expressing it in cells can all be performed using conventional techniques (Reference [Gene Manipulation Manual]
Edited by Yasutaka Takagi, Kodansha (1982); Maniat
is et al, “Mo1ecular Clo
ning: A LaboratoryManual
” Co1d Spring Harbor Labo
ratory, Co1d Spring Har b
or, New York (1982);
rook et al, “Mo1ecular Cl
oning: A Laboratory Manua
l 2nd Ed,” Co1d Spring Ha
rbor Laboratory, Cold Spr.
ing Harbor, New York (198
9)). [0014] [Example 1] Obtaining human thrombomodulin gene a)
Probe creation literature (J, Biochem, 103.281-285
(1988)) synthesized based on the DNA sequence described in
A probe was created using NA oligomer. That is, P
r-TM-01: and Pr-TV-02, which forms a partially complementary chain thereto, were synthesized. This is a portion corresponding to 13 bases downstream of the stop codon of the human thrombomodulin gene. After annealing these two synthetic NA oligomers to form a partial double strand, dNTPs
Complete double strands were obtained by treatment with T4 DNA polymerase in the presence of T4 DNA polymerase. After phosphorylating the 5' end of this DNA fragment with T4 polynucleotide kinase, it was ligated with pUc119 (commercially available) cut with HincII using T4 DNA ligase, resulting in a plasmid pUCp r TM9 in which three of the former were repeatedly inserted in the same direction. I got it. This pUCp
rTM9 was cut with EcoRI and HindIII to separate the synthetic oligomer-derived portion, labeled with 32p by the nick translation method, and used as a probe for obtaining the human thrombomodulin gene. [0015] b) Cloning of human thrombomodulin gene Human chromosomal DNA was partially digested with Alu I and Hael II to create a human gene library (vector was Cha
ron 4A) and from this library, a)
The human thrombomodulin gene was screened by plaque hybridization using the probe prepared by the method described in (Reference: "Gene Manipulation Manual", edited by Yasutaka Takagi, Kodansha (1982)). As a result, DNA of a phage clone (phage No. 7) retaining the full length of the human thrombomodulin gene was obtained. This DNA was cut with Sac I to isolate a 6.6 kbp DNA fragment containing the human thrombomodulin gene. This DNA fragment was inserted into the Sac I site of pUc119 to obtain plasmid p7TM-5ac I shown in the upper row of FIG. In the figure, TM indicates the human thrombomodulin gene region. This p7TM-5ac I was combined with Xbo I and N
After cutting with coI and making blunt ends using the Klenow fragment, the 2, IKbp DNA fragment containing the human thrombomodulin gene was separated and inserted into the HincII site of pUc119.
Plasmid p7TMO1 inserted in the direction of II side
was obtained (lower row of Figure 1). The single-stranded DNA of this plasmid was prepared and the DNA base sequence was examined using the dideoxy method.
(1988)) was confirmed to match the DNA sequence of the human thrombomodulin gene. [0016]

[Example 2] Construction of expression vector As shown in Figure 2, plasmid p7TMO1 was transformed into 5phI
and PvuII, and a 470 bp DNA fragment containing the start codon ATG of the human thrombomodulin gene was isolated. After cutting this fragment with Ban I, Kleno
w fragment to make blunt ends, and then BgllI
The tsobp DNA fragment (fragment A) containing the ATG codon was isolated. On the other hand, plasmid p7TM01
was cut with 5phI, blunt-ended using mung bean nuclease, and cut with BglII to isolate a 4 to 8 kbp DNA fragment containing a portion of the vector. this DNA
The fragment and the above fragment A were ligated using T4 DNA ligase to produce plasmid p7TM17. This is p7
It is obtained by removing most of the 5' non-coding region of the human thrombomodulin gene from TMO1. Note that Hi on the 5' side
The remaining non-coding region up to the ndll1 site is about 30 bp. (00171) Next, a terminator sequence was ligated to this full-length human thrombomodulin gene. The SV2 terminator of the plasmid psV2-gpt (commercially available) was used for the terminator sequence.
After cutting t with Apa I and BamHI, Kle
The now fragment was used to make blunt ends, and an 850 bp DNA fragment containing the SV2 transcription termination region was isolated. This fragment was inserted into the vector pUc19, which had been cut with Sat I and blunt-ended with the Klenow fragment. A plasmid inserted in such a direction that the 5' side of the transcription termination region was on the EcoRI side of pUc19 was taken and designated as psVTOl. Plasmid psVTO1 was combined with BamHI and 1nd
After cutting with llI, the ends were made blunt with mung bean nuclease, and an 850 bp DNA fragment containing the transcription termination region was isolated. The above fragment of the transcription termination region was inserted into p7TMi7 which had been cut with BstXI and Xba I and blunt-ended with mung bean nuclease. A plasmid in which the human thrombomodulin gene and the transcription termination region are in the same direction was taken and designated p7TM19. [00181 Next, partial-length human thrombomodulin DNA
A vector was created. As shown in FIG. 4, plasmid p7TM19 was cleaved with Nru I, treated with Bal31S, further treated with Xba I, and then blunt-ended with Klenow fragment. By self-ligating this with T4 DNA ligase, plasmid pTM
Got s07. l) Human thrombomodulin encoded by the gene held by TMs07 is from the 1st alanine to the 491st alanine (the amino acid numbers are from the document EMBOJ, 6.1891-1897)
(1987)). [0019] Next, the promoter region of the marker gene was constructed (FIGS. 5 to 7). The plasmid SV2-gpt containing the SV2 promoter and SV2 terminator was transformed into Eco
After cutting with RI and Pvull, the ends were made blunt with Klenow fragment, a 2.9 kbp DNA fragment containing the xanthine-guanine phospholiposyl transferase (GPT) gene was taken, and this was inserted into the HinclI site of pUc13. SV2 promoter is pU
Take the plasmid inserted into the EcoRI side of c13 and p
DA I-gpt (Fig. 5), HindIII
The one inserted on the side was designated pNAN-gpt (Fig. 7). After cutting the plasmid psV2-gpt with EcoRI and PvulI, a 2.9 kbp DNA containing gpt was obtained.
The A fragment was isolated and ligated with pUc13 cut with EcoRI and SmaI. The obtained plasmid is pT
EN-gpt (Fig. 6). pDAI-gpt
After cutting with indIII and making blunt ends with the Klenow fragment, the 3.0 kbp containing the SV2 promoter (
7) The DNA fragment (fragment B) was isolated. pDAI-gl
) After cutting t with BglII and BamHI, Kleno
D containing the gptm region after blunt-ending with w fragment
The NA fragment (1,8 kbp) was isolated and combined with fragment B. The plasmid ligated in the direction in which the gpt gene is expressed was taken and sealed into pD-gptB-84 (Figure 5).
pD-gptB-84 was cut with Xba I and EcoRV to create 0.8 kbp DNA containing the SV2 promoter.
The fragment was isolated and pTEN-gp was cut with Xba I and EcoRV to remove the part containing the SV2 promoter.
Inserted into t. The obtained plasmid was transformed into pT-gpt
B-23 (Figure 6). This pT-gp tB-23
was cut with Hindll and EcoRV to isolate a 0.8 kbp DNA fragment containing the SV2 promoter, and
It was inserted into pNAN-gpt which had been cut with ndIII and EcoRV to remove the SV2 promoter portion. The obtained plasmid was named pN-gptB-16 (
Figure 7). (The neomycin resistance gene (neor) was used as the 00201 marker gene. As shown in FIG. 8, plasmid pSV2-neo (commercially available) was cut with BglII and BamHI to contain the neomycin resistance gene.
A 3 kbp DNA fragment (fragment C) was isolated. pN-g
After cutting p tB-16 with BgllI and BamHI, the SV2 promoter and ampicillin resistance gene (AI
npr) was separated and combined with fragment C to obtain plasmid pB-neo. [0021] pB-neo with XbaI and BamHI
and 2. containing the neomycin resistance gene after cutting with Sca I and making blunt ends with mung bean nuclease.
A DNA fragment of 7 kbl) was isolated (Fig. 9). This DN
The A fragment was ligated with pTMs07, which had been cut with BamHI and made blunt-ended with a KIenOW7 fragment, to obtain plasmid pTMs07-neo in which the human thrombomodulin gene and neomycin resistance gene were inserted in the same direction. on the other hand. pO-gal (Literature DNA, 8,127-133(
(1989)) was cut with HindlII to isolate 0.5kbp DNA containing the R5V promoter, and this 0.5kbp DNA
pTMs07-n in which the bp fragment was cut with Hindll
Combined with eo. A plasmid in which the promoter was inserted in a direction that allowed expression of the human thrombomodulin gene was selected and designated pRS7TMneo. This plasmid p
E, col iR57T with RS7TM-neo
M-neo has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (accession number: FER No. 2609, FER
M BP-2609, Deposit: September 25, 1989). [0022]

[Example 3] Establishment of expression cell line 20 µg of pR37TM-neo was added to 440 µl of water, and then 60 µm of 2MCaCl2 solution was added to make one solution. XZHBS liquid (HEPE52.5g, NaC13
,2g/200ml; pH7゜1) 500μl of ×100 phosphoric acid solution (Na2HPO41,253g,
NaH2PO40,546g/100ml) 10 l
The mixture containing Ll was made into a 2nd solution. The first liquid was added little by little to the second liquid while stirring, and the mixture was left at room temperature for 30 minutes. On the other hand, 10
1 to 1 in Dulbecco's modified Eagle medium "Nissui" ■ (Nippon Pharmaceutical, hereinafter referred to as DME■ medium) supplemented with % FBS.
2 x 105 CHO- to 1 cell (Dainippon Pharmaceutical Co., Ltd.)
Catalog number: 03-402. Original ATCC No.: C
CL-61) was cultured overnight in a 75 cm2 culture bottle. Four hours after replacing the medium with 10 ml of the same fresh medium, the above plasmid suspension was added and cultured for 18 hours. DME containing 1 g/l G418 (manufactured by GIBCO)
The culture medium was replaced with 16 ml and culture was continued. After culturing for 10 days while changing the medium every 3 to 4 days, transformed strains were selected by limiting dilution method to obtain human thrombomodulin producing strain CHO-KIR87TMneo No. 2.
-9b-29 (hereinafter abbreviated as No. 2-9b-29) was obtained. [0023]

[Example 4] Quantification of human thrombomodulin by measuring the activity of activated protein C (APC method) 33 μl of gelatin buffer (0.1% gelatin (manufactured by SIGMA),
Catalog number G-2500), 20mM Tris
-HCI (manufactured by SIGMA), 0.1M NaC1
, 0,02% NaN 3 ; 1) H7,5) and 50
6 μl of mM CaC1z, 3 μM human protein C (
American Diagnostical C6
Co., Ltd.) and 1 μl of the measurement sample were mixed and allowed to stand at 37° C. for 20 minutes. This was supplemented with 3.5 U/ml bovine thrombin (manufactured by Kaori Seiyaku; 5mM MES, 0.1M
NaCl, 0.02% NaN3. (Soluble at pH 6.0)
After adding 10 μl, leave it at 37°C for 10 minutes, then add 20 μl.
antithrombin III (Neuert 500x (
(manufactured by Green Cross) dissolved in 20 ml of physiological saline) and 20 μl of heparin (manufactured by SIGMA, catalog number H-3125; 10.000 u dissolved in 25 ml of gelatin buffer) are added. After mixing thoroughly, transfer 10 μl to another container and add 90 μl of S-2.
366 (Daiichi Kagaku; dissolved in gelatin buffer and 0.1
Vmax (Molecular
405n per unit time generated by the activated protein C produced here.
Human thrombomodulin in the measurement sample is quantified by measuring the change in absorbance at m. [0024]

[Example 5] Human thrombomodulin-producing CHO cell line No. 2-9b-29 obtained in Example 3 was incubated with 2×10 cells per cell, GIT medium containing η18 (Ig/l) and aprotinin (50 U/ml) ( Made by Nippon Pharmaceutical)
Inoculated into a roller bottle containing 200ml, 0.3~
Culture was performed at 0.5 rpm. On the second day, the medium was replaced with the above medium. On the 4th day, the medium was replaced with a medium in which 6418 was removed from the above medium. Thereafter, replace the medium every day until the 7th day, and
．． The culture fluid collected on the 6th and 7th day was combined and used as the material for product collection. [0025]

[Example 6] Culture solution obtained in Example 5 (880ml)
After removing solid impurities from 1. by centrifugation (3000 rpm, 10 minutes) and filtration (using 0.8 μn+ membrane filter). OM Tris-HC
I buffer (pH 7.5) was added to adjust the pH to 7.5. This solution was mixed with 20mM containing 0.15M NaCl.
Q-Sepharose Fast Flow (Pharma) pre-equilibrated with Tris-HCI buffer (pH 7,5)
The mixture was passed through a packed column (manufactured by CIA) (medium 2°5 x 12 cm) at a flow rate of 200 ml/hour. After washing with 300 ml of the above buffer solution, the column flow rate was 20 ml/h.
'20mM Tris-HCI buffer (pH 7,5)
Elution was carried out using a linear sodium chloride concentration gradient from 0.15M to 1.20M (total elution buffer volume was 10010
0O. The eluate was fractionated into 19 ml portions, and each fraction was examined for activity using the APC method. As a result, two activity peaks were detected in fractions N008-15 and 22-32 (see FIG. 10). Collect the latter peak fraction and fraction A
And so. Both of the former fractions were designated as fraction B. [0026]

[Example 7] Both components A and B were further purified by affinity chromatography. 0.15M NaCl in advance
20mM Tris-HCI buffer (pH 7,5
) and anti-human thrombomodulin IgG-conjugated cellulofine (formylcellulofine (manufactured by Chisso)).
anti-human thrombomodulin IgG bound at a ratio of approximately 5 μm/ml gel) packed column (φ1.5×6c)
m), double the volume of 20mM Tris-HCI buffer (
Fraction A (or B) diluted with pH 7.5) at a flow rate of 20 m
It passed at l/hour. This column was loaded with 0.35M NaCl at a flow rate of 30ml/hour.
20mM Tris-HCl buffer (pH 7,
After washing with 5), elution was carried out with 150 ml of 20 ml Tris-HCI buffer (pH 7, 5) containing 3.0 M potassium thiocyanate at a flow rate of 30 ml/h. This eluate was collected and filtered using an ultrafiltration membrane (diaflow membrane YM30).
It was concentrated to about 5 ml using an ultrafiltration device equipped with a filter (φ76 mm). Add to this 20 ml Tris-HCI buffer (pH 7) containing 100 ml of 0.15 M NaCl.
, 5) was added and concentrated again to about 5 ml in the same manner. After repeating this operation twice, the solution was finally concentrated to 1 ml using YM30 with a diameter of 43 mm. [0027] The active substance from fraction A thus obtained was TM
-β, the active substance from fraction B was designated as TM-α. Reverse phase HPLC was performed on each sample. The elution conditions are as follows. The results are shown in FIG. TM-α showed a single peak (Figure 11 (A)), but TM-β was not eluted under such acidic elution conditions (Figure 11 (B)).
. [0028] Therefore, tPLc analysis was performed by changing the elution conditions to alkaline conditions as described below. As shown in FIG. 12, under these conditions, 1-β was eluted as a single peak at a retention time of 13.0 minutes, and 1-β could be purified. Both purified TM-α and TM-β showed a single band in 5DS-polyacrylamide electrophoresis. [002g1 Regarding TM-α and TM-β, the amino acid sequences on the N-terminal side were analyzed using a peptide sequencer (Ap
manufactured by plied Biosystems, type 470A)
When I investigated up to the 5th place using
It was ro-Ala-61u-Pro. This is from the literature (EMBOJ, 6.1891-1897 (1987
) The N-terminal amino acid sequence of human thrombomodulin is identical to that described in ). From the difference in HPLC behavior between TM-α and TM-β, it was inferred that resistant-β was due to the addition of acidic sugar chains. [00301 FIG. 13 shows the elution pattern of ion exchange chromatography of TM-β. The elution conditions are as follows. [0031]

[Example 8] Ta1l-chondroitinase AB
Although not known for C-treated human thrombomodulin,
A portion of rabbit thrombomodulin is modified with chondroitin sulfate-like/dermatan sulfate-like glycosaminoglycans, and it has been reported that it exhibits antithrombin III-dependent antithrombin activity through the glycosaminoglycan moiety. , the importance of sulfated glycosaminoglycans has been shown (J, Biol, Chem, 2
63.8044-8052 (1988): Throm
b, Res, 54.27-39 (1989))
Therefore, the presence or absence of sulfated glycosaminoglycan was investigated for TM-β, which is an acidic substance. First, TM-β was treated with chondroitinase ABC, which specifically decomposes chondroitin sulfate and dermatan sulfate to produce sulfated unsaturated trisaccharides. Chondroitinase ABC (protease free, IU/vial; manufactured by Seikagaku Corporation) was added to a vial containing lyophilized powder with 620 μl of 50 mM Tris-HCI buffer (pH 8,0) containing 0.1 M NaCl and 3
0 μl of 1.0 M sodium acetate aqueous solution was added to dissolve the enzyme. To this, 350 μl of TM-β solution (3.4 m
g/ml, 0. LM NaCl-20mM Tri
After adding s-HCI buffer (pH 7.5) and stirring well, the mixture was reacted at 37°C for 16 hours. TM-β was quantified using ELI using lyophilized powder of TM-α as a standard sample.
SA(enzyme-11nke d 1nuaunO
5Orvent assay method). At this time, the weight conversion is based on the mobility of 5DS-electrophoresis, T
The molecular weight of M-α is 70 kD, and the molecular weight of TM-β is 8.
It was performed as 5kD. [0032] [Example 9] TSKgel Phenyl 5P attached to an HPLC device (LC-6AD, manufactured by Takasugi Seisakusho)
W RP (φ4.6 x 75mm; manufactured by Tosoh Corporation)
10% CH in advance at a flow rate of 1.0 ml/min.
Equilibrate with 1mM NH4OH solution containing 3CN,
Under these conditions, the reaction solution obtained in Example 8 was injected into the column that was being continuously passed through the column. By this operation, most of the proteins are adsorbed on the column, but the unsaturated trisaccharide, which is a reaction product, passes through without being adsorbed. This pass-through fraction was collected and lyophilized, the lyophilized powder was dissolved in 1.0 ml of water, and HPL
It was used as a sample for identification by C. [0033] Example 10 The sample obtained in Example 9 was analyzed by HPLC under the following conditions. Under these conditions, the retention time of the main peak in the sample was 24.
It took 7 minutes (Figure 14). For convenience, the unsaturated trisaccharide exhibiting this peak is shown in the drawing as ΔDi-XS. This retention time was determined using commercially available standard 2-acetamide-2-deoxy.
-3-0-(β-D-gluco-4-enepyra
nosyluronic acid)-4-0-sulf
This coincided with the retention time of o-D-galactose (hereinafter abbreviated as ΔDi-4S; manufactured by Seikagaku Corporation). Furthermore, when this sample (ΔDi-XS) and ΔD 1-4S were mixed and analyzed under the above conditions, a corresponding increase was observed in the main peak (retention time 24.7 minutes) in the sample (Fig. 1
5). ΔDi-45 is known to be a degradation product when chondroitin-4-sulfate or dermatan sulfate is treated with chondroitinase ABC, and the main modified sugar chain of TM-β is chondroitin-4-sulfate or dermatan sulfate. I could guess that it was. Note that the peak at a retention time of 16.1 minutes in FIG. 14.15 is a peak due to 5CN- ions mixed in the sample (mixed by the operation in Example 7). [0034]

Example 11 The sample obtained in Example 9 was analyzed by HPLC under the following conditions different from those in Example 10. Even under these conditions, the retention time of the main peak in the sample (21,
3 minutes) was consistent with the retention time of the predicted degradation product ΔDi-45 (not shown). [0035]

[Example 12] Next, in order to clarify whether the TM-β-modified sugar chain is chondroitin-4-sulfate or dermatan sulfate, chondroitinase ACI Flavo (which specifically decomposes chondroitin-4-sulfate) produces sulfated unsaturated trisaccharides, but cannot decompose dermatan sulfate) and T
M-β was treated. 50 μl of TM-β solution (3,4 m
g/ml, o, 15M NaCl20mM Tri
s-HCI buffer (pH 7,5)) with 50 μl of 0.
4M TriS-HC1 buffer (pH 7,5) and 50
μm of 0.4 M sodium acetate solution and 50 μl of 0.1
% BSA solution and 200 μl of water, and to this add commercially available chondroitinase ACI flavo (1.6 U/ml pre-dissolved in 0.1% BSA solution at a concentration of IU, ml).
After adding 100 LL1 of the vial (manufactured by Seikagaku Corporation) solution, the mixture was stirred well and reacted at 37° C. for 6 hours. From the obtained reaction solution, the unsaturated trisaccharide fraction was collected in the same manner as in Example 9,
When subjected to HPLC under the same conditions as in Example 10.11, the predicted decomposition product Δ was observed in both cases as in Examples 10 and 11.
A peak with the same retention time as Di-45 was observed. FIG. 16 shows the HPLC elution pattern under the same conditions as in Example 10. Therefore, it was inferred that the modified sugar chain of TM-β was chondroitin-4-sulfate. [0036]

[Example 13] In order to further confirm that the modified sugar chain of TM-β is chondroitin-4-sulfate,
ΔDi-XS was treated with chondro-4-sulfatase, which specifically acts on the predicted degradation product ΔDi-45 and desulfates it. Add 10 μl of 0 to the 50 μm sample obtained in Example 9.
．． 4M Tris-HCI buffer (pH 7,5) and 1
0 μl of 0.4 M sodium acetate solution and 10 μl of 0.4 M sodium acetate solution.
Add 1% BSA solution and 10 μl of water;
l of commercially available chondro 4-sulfatase (IU/ml
After adding O, 1% BSA (manufactured by Seikagaku Corporation) and stirring well, the mixture was reacted at 37° C. for 1 hour. Immediately after the reaction was stopped by heating at 100° C. for 2 minutes, analysis using HPLC was conducted under the conditions described in Example 10. As a result, the peak with a retention time of 24.7 minutes decreased and a new peak with a retention time of 15.
A peak of 6 minutes was observed (Figure 17(C)''), and this retention time was lower than that of the commercially available standard 2-acetamido-2-
deoxy-3-0-(β-D-gluco-4-en
epyranosyl uronic acid)-D
-galactose (hereinafter abbreviated as ΔDi-O8)
(Figure 17(A)), and commercially available ΔDi
-45 and 2-acetamido-2-deoxy-3
-0-(β-D-gluco-4-enepyrano
sylur acid)-6-0-sulf
2.5 μg each of o-D-galactose (hereinafter abbreviated as ΔDi-65: manufactured by Seikagaku Corporation) was added to Chondro 4.
- treated with sulfatase and analyzed by HPLC under the same conditions. As a result, in ΔDi-43, the retention time was Δ
It produced a peak equal to that of Di-OS (Figure 17 (B)
). However, no peak shift was observed in ΔDi-6S, which is known as a degradation product of chondroitin-6-sulfate, due to chondroitin-4-sulfatase treatment (FIG. 17(D)). The above results revealed that the recombinant human thrombomodulin (TM-β) of the present invention has a sulfated glycosaminoglycan based on chondroitin-4-sulfate. In addition, as a result of converting the amount of ΔDi-45 produced from the peak area in HPLC, the above ΔDi of 20 to 25 molecules per molecule from recombinant human thrombomodulin was obtained.
It was assumed that -45 was produced by chondroitinase treatment. [0037]

[Example 14] Measurement of anticoagulant activity of TV-54 nM bovine thrombin (abbreviated as T in Table 1) in physiological saline solution (
(manufactured by Kaori Pharmaceutical) 100μl, bovine fibrinogen (type 2; 3mg/'ml, 20mM Tris-HC)
I, 0.15M NaCl pH 7°5; manufactured by Dai-Kagakuyakuhin) 100 μl and 0.9% NaCl 1.1 m
g/ml BSA, 0154.108 or 162 nM TM-β100 dissolved in 0.1% Lubrol px
The reaction was started by mixing μm, and the time until solidification was measured. The measurement was performed using an automatic blood coagulation measuring device Crotic II (manufactured by Metek). The results are as follows. The numerical unit is seconds. [0038] [Table 1] [00391] As shown in Table 00391, human thrombomodulin having a sulfated glycosaminoglycan chain, TM-β, inhibited the coagulation activity of thrombin. [00401

[Example 15] Promotion of protein C activation of thrombin by TM-β 20.15.8.6.4.2 and ][g T
An M-β solution was prepared, and activated protein C was produced using the method of Example 4 using this as a measurement sample. The absorbance per minute (OD) caused by the generated activated protein C
4 o = ) changes are shown in FIG. As shown in the figure,
TM-β, a human thrombomodulin with sulfated glycosaminoglycan chains, promoted protein C activation by thrombin. [0041] [Example 16] TM treated with chondroitinase ABC
Blood concentration half-life of -β TM-β was treated with chondroitinase ABC in the same manner as in Example 8, injected into an HPLC column in the same manner as in Example 9, and the column was thoroughly washed to remove the reaction product. Unsaturated trisaccharides were removed. Next, chondroitinase ABC-treated TM-β adsorbed and retained on the column was eluted with 1 mM NH40H-acetonitrile (5-65% linear concentration gradient).
This was collected and freeze-dried. This freeze-dried sample was 0.9
%NaC1,0,1% Lubrol PX, 1■/ml
It was dissolved in BSA and used in the following experiment. [00421 Wistar male rat (Shizuoka Experimental Animals)
At 11 weeks of age, chondroitinase ABC was administered under Bendoparbital (trade name, Nembutal, manufactured by Dainippon Pharmaceutical Co., Ltd.) anesthesia.
Treated TM-β was administered via the femoral vein at a dose of 0.2 μg/kg. After administration, 5.10.30.60.120.30
Add citric acid to blood (3.13% sodium citrate/
Dihydrate:blood = l:9) was collected and centrifuged (3,000
0 rpm, 10 minutes) to obtain plasma. This plasma is P
Blood chondroitinase ABC was determined by ELISA by diluting 100 times with BS (containing 0.02% Lubrol px).
The amount of treated 1-β was quantified. Conversion of TM-β amount is as per Example 8
Similarly, TM-α freeze-dried powder was used as a standard sample. [00431 As shown in FIG. 19, the blood concentration half-life of chondroitinase ABC-treated TM-β(-.-) is 7.
7 hours, which was significantly longer than the blood concentration half-life of about 20 minutes in the case of untreated TM-β (-〇-). Note that chondroitinase ABC-treated TM-β did not lose its ability to promote protein C activation by thrombin. Therefore, if a human thrombomodulin derivative that is not modified with sulfated glycosaminoglycan chains can be produced, it can be expected that recombinant human thrombomodulin will have a longer half-life in blood than TM-β. [00441

[Example 17] Creation of human thrombomodulin derivative gene and construction of its expression vector Plasmid pTMs07 containing the partial length human thrombomodulin gene was cut with Bs5HII and NheI, then blunt-ended with the Klenow fragment, and self-
Bs5HI of approximately 1.3 kbp
Plasmid pTMDs with I-NheI fragment removed
07 (upper row of FIG. 20). Separate DNA oligomer Mu
t-TMSG-2: (5')GCTCGCCAGA
Synthesize GTCGCCACCG(3”) and Kunk
eI method (References: Sambrook et al.
: ”Mo1ecula r Cloning: A
Laboratory Manual, 2nd Ed
,” Vol, 2. p15.74. Cold S
pringHarbor Laboratory, C
o1d Spring Harbor, New Yo
Mutations were introduced into pTMDs07 using a site-directed mutagenesis method according to M.R.K. (1989). For experiments, use the site-directed mutation experiment kit rMutan"-K" (
(manufactured by Takara Shuzo) was used. That is, the outline is as follows (Figure 20.21). [004511) Obtaining 5sDNA containing dU pTMD
s07 was maintained in E. coli MV1184, and the bacteria were cultured in 2×YT medium (containing 10,+1 g/ml tetracycline and 30 μg/ml streptomycin).
precultured with Add 30 μl of this culture solution to 2XYT medium (1
(containing 50 μg/ml of ampicillin) and inoculate 3 ml of phage M13KO7 with m, o, i, =
2 to 10, and after standing at 37℃ for 30 minutes, 70μg/
Kanamycin was added to the mixture to give a total volume of 1.0 ml, and cultured with shaking at 37° C. overnight. The supernatant was collected by centrifugation and filtered through a 0.22 μm membrane filter, and then 20 μl of this supernatant was mixed with 80 μl of E. coli BW313 culture solution.
After standing at 7°C for 10 minutes, an appropriate amount was placed on an LB-plate (150μ
(containing g/ml ampicillin) and allowed to form colonies at 37°C. A single colony was precultured in 2XYT medium (containing 150 μg/ml ampicillin), and 1 ml of this culture was incubated with 2XYT medium (150 μg/ml).
of ampicillin) and infected with phage M13KO7 at m, o, i = 2 to 10. After standing at 37°C for 30 minutes, kanamycin was added at a concentration of 70 μg/ml, and cultured overnight at 37°C with shaking. The supernatant was collected by centrifugation. 20% PEG6000/2 in supernatant
．． 25 ml of 5M NaCl solution was added and stirred, and after being left at room temperature for 10 minutes, the precipitate was collected by centrifugation. TE buffer 5u
Add an equal amount of neutralized phenol dissolved in +l and stir for 10 minutes.
I left it for a minute. The aqueous layer was collected by centrifugation, and an equal amount of neutralized phenol:chloroform:isoamyl alcohol (25:
24:1) was added, stirred, and then allowed to stand for 10 minutes. The aqueous layer was collected by centrifugation, and an equal amount of chloroform:isoamyl alcohol (24:1) was added, stirred, and then allowed to stand for 10 minutes. After centrifugation, collect the aqueous layer and add 3M ammonium acetate,
500ul of pH 8.0, 5ml of isopropyl alcohol
After stirring, centrifugation was performed to collect the precipitate. The precipitate was washed with 70% ethanol, dried under reduced pressure, and then
Dissolved in E buffer. [0046] if) Introduction of site-specific mutations 10 pmo
1 of the above synthetic oligomer Mut-TMSG-2 was AT
The 5' end was phosphorylated by T4 polynucleotide kinase in the presence of P (final volume of reaction solution was 10 μl). 1 μl of this solution and the 5sDNA solution obtained in i) (0.2p
Mix 1 μl of mol/10 μl annealing buffer) and
Hybridization was carried out by standing at 5°C for 15 minutes and at 37°C. E, colt DNA ligase, and T4 DNA polymerase were added to this in the presence of dNTPs (reaction solution 2).
7 μl) After standing at 25°C for 2 hours, add 3 μl of 0.2M ED.
TA, pH 8.0 was added and left at 65°C for 5 minutes to stop the reaction. 3 μl of this reaction solution was added to E, cot i BMH
71-18mutS Mixed with competent cells and 0℃3
0 minutes, 45 seconds at 42°C, and 1 to 2 minutes at 0°C, then SOC
300 μl of culture medium was added and cultured with shaking at 37° C. for 1 hour. This was infected with M13KO7 phage, left standing at 37°C for 30 minutes, 1 ml of 2XYT medium (containing 150 μg/ml of ampicillin and 70 μg/ml of kanamycin) was added, and cultured with shaking at 37°C overnight. 20 μm of supernatant collected by centrifugation was transferred to 80 μm of E. coli MV1184 culture.
After mixing and leaving at 37℃ for 10 minutes, transfer to LB-plate (150℃).
Containing μg/ml ampicillin), colonies were grown at 37°C. From this, a plasmid pM2TMDO7 into which a partially specific mutation had been introduced was obtained (Figure 20, bottom row).
. [0047] pM2TMDO7 with Nhel and 5al
After cutting with I, a DNA fragment of approximately 260 bp containing the mutation introduction site was isolated. This fragment was transformed into pRS7TM-neo
Vector pR37M2TM for expressing a defective mutant human thrombomodulin gene by combining it with a DNA fragment containing a promoter separated after cutting with el and 5alI
-neo was created (Figure 21). E. coli carrying this plasmid vector pRS7M2TM-neo
R57M2TM-neo has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Accession number: FIKEN Article No. 317).
No. 7). [0048] [Example 18] In the same manner as in Example 3, pRS7M2
Transform CHO-Kl cells using TM-neo,
Recombination defective mutant human thrombomodulin derivative (hereinafter M
Expression cell line CHO-KIR37M (referred to as TMIO)
2TM neo No. 14-50 (hereinafter referred to as No.
14-50) was obtained. [00491

[Example 19] In the same manner as in Example 5, MTMIO production cell line No. 14-50 was cultured in a roller bottle, and the culture fluid collected on day 5.6 and 7 was combined and used as material for product recovery. did. [Example 20] The culture solution (4 L) obtained in Example 19 was centrifuged (3,000 rpm, 10 minutes) and filtered (
After removing solid impurities by treatment (using a 0.45 μm membrane filter), 1. OM Tris-HC
I) buffer (pH 7.5) was added to adjust I)H to 7.5. Add this solution to 20mM containing 0.15M NaCl.
Q-Sepharose Fast Flow (Pharma) pre-equilibrated with Tr 1s-HCI buffer (pH 7,5)
C ia) packed column (φ5 x 10 cm) at a flow rate of 3.
It passed through at 0 m1/min. After washing with 200 ml of the above buffer at a column flow rate of 20 ml/min, the column flow rate was 20 ml/min.
．． Elution was performed with 600 ml of 20 mM Tris-HCI buffer (pH 7,5) containing 5M NaCl. When MTMIO in the elution fraction was quantified by ELISA (Sand-Deutsch method using two types of anti-human thrombomodulin monoclonal antibodies), the recovery rate was 99%. [00511

[Example 21] The active fraction of Example 20 was purified by affinity chromatography. 0.15M NaCl in advance
Anti-human thrombomodulin IgG was bound to anti-human thrombomodulin IgG-conjugated cellulofine (formylcellulofine (manufactured by Chisso) at a ratio of approximately 5 μm/ml gel) containing 20 mM Tris-HCI buffer (pH 7.5). packed column (medium 2゜5 x 9cm)
) at a flow rate of 60 ml/h. This column was loaded with 0.35M NaCl at a flow rate of 60ml/h.
20mM Tris-HCI buffer (pH 7,5) containing
20mM Tris-HCI buffer (pH 7°5) containing sodium thiocyanate (60ml/h)
It was eluted with 400ml. Collect this eluate and use an ultrafiltration membrane (
It was concentrated to about 5 ml using an ultrafiltration device equipped with a diaflow membrane (YM30, φ76 mm). 100 for this
m1 of 20mM Tris- containing 0.15M NaCl
Add HCI buffer (pH 7.5) and incubate again in the same way for about 5.
Concentrated to ml. After repeating this operation further, φ
Finally 22m using 43mm ultrafiltration membrane YM30
It was concentrated to 1. [0052] [Example 22] Total 5 obtained as in Example 21
0 tons of MTMIO fractions (total volume approximately 10 tons)
0ml) Gel filtration chromatography was performed. PBS (KCI 200μ in ll, KH2PO4
200mg of NaCl, 8g of Na2PO4・
The MTMIO fraction was passed through a Sephacryl S-35-300HR (manufactured by Pharia) packed column (medium 5° OX 95 cm) equilibrated with 7H20 (containing 2.16 g of 7H20) at a flow rate of 5 ml/min. P for column flow rate 5m1Z
Elution was performed with BS, and the eluate was fractionated into 15 ml portions. Collect fractions from 41st to 47th and make 40
It was concentrated to ml. The thus obtained purified MTMIO was subjected to reverse phase HPLC in the same manner as in Example 7. The elution conditions are as follows. As shown in FIG. 22, MTMIO showed a single peak. Regarding MTMIO, the amino acid sequence on the N-terminal side was determined using a peptide sequencer (Applied Bi
When I investigated up to the 5th item using a 470A model manufactured by OSYSTEMS, the main ones were Ala・Pro-Ala-
It was Glu-Pro. This is the literature (E!1lBOJ
The amino acid sequence corresponds to the N-terminal amino acid sequence of human thrombomodulin described in 1987). [0053]

[Example 23] Chondroitinase ABC of MTMIO
It was investigated whether treated MTMIO retained chondroitin sulfate. 620 μl of 1.0 M NaC in a vial containing lyophilized chondroitinase ABC (protease free, IU/vial; Seikagaku Corporation)
1 in 50mM Tris-HCI buffer (pH 8
, 0) and 30 μm of O, 1M aqueous sodium acetate solution was added to dissolve the enzyme. To this, 350μI of MTMIO solution (5,0mg/n+l, 0.1M NaCNaC1
-2OTris-HCI buffer (pH 7,5))
After stirring well, the mixture was reacted at 37°C for 16 hours. When the reaction product and unreacted MTMIO were subjected to reverse phase HPLC under the same conditions as in Example 7, the retention times of both were found to be the same (not shown). This indicates that unlike TM-β, MTMIO is not modified with glycosaminoglycan that is cleaved by chondroitinase ABC. [0054] [Example 24] Blood half-life of MTMIO: Wistar rats (male, approximately 250 g) aged 9 to 10 weeks were anesthetized with bendoparbital, and MTMIO was administered at a dose of 1. It was administered from Blood was collected over time, Example 16
The residual amount in blood was measured by ELISA method in the same manner as above. As a result, the blood half-life of MTMIO was approximately 7 hours (Figure 2
3). As described above, the derivative MTMIO, which was created by changing the amino acid sequence of the site where chondroitin sulfate that modifies TM-β is predicted to bind, has a sulfuric acid sequence that is cleaved by chondroitinase ABC. It is thought to have a long half-life in the blood because it is not modified with glycosaminoglycans. [0055] [Example 25] Ability to promote protein C activation of thrombin by MTMIO 40.20.15.10 and 5 nM MTMIO solutions were prepared and used as measurement samples. This measurement sample is 6 μm
and 32 μl of Buffer A (20 mM Tris-HC
I (manufactured by SIGMA), 0.15M NaC1,0,5
%BSA (manufactured by SIGMA, catalog number A-4378
) ;I)H7,4) , 50mM CaC1z 6
μm, 3 μM human protein C (American
DiagnO3tica Inc.) 10μl
mixed with. To this, 100nli human thrombin (S
After adding 6 μl (manufactured by IGMA, catalog number T-3010; dissolved in buffer A), let stand at 37°C for 15 minutes, and add 20 μl.
antithrombin III (Neuert 500x (
After dissolving Buffer B (50mM Tris-HCI (SIGMA)) in 20ml of physiological saline,
), 0.1M NaCl, 1mM CaCl2;
pH 8,0) diluted 2.5 times) and 20 μl
Heparin solution (manufactured by SIGMA, catalog number H-3
125: Gelatin buffer (0.1% gelatin (manufactured by SIGMA, catalog number G-2500), 20mM Tris-HCI, 0.1% gelatin to give a concentration of 400u/ml), 20mM Tris-HCI,
IM NaCl, 0,02% NaN3: pH 7,5)
(adjusted concentration) was added. After mixing thoroughly, 100 μl of S-2366 (adjusted to 2.0 mM with No. 1 Chemical Digelatin Buffer and then 0.4 mM with Buffer B) was added to Vmax (Molecular D
405 nm per unit time caused by the activated protein C produced here.
M in the measurement sample by measuring the change in absorbance at
The coenzyme activity of TMIO was investigated. Absorbance per minute caused by the generated activated protein C (○D40
5) is shown in FIG. 24. As shown in the figure, MTMI
O promoted protein C activation by thrombin. [0056] [Example 26] Measurement of anticoagulant activity of MTMIO 54n
M bovine thrombin (abbreviated as T in Table 2) in physiological saline solution (manufactured by Kaori Pharmaceutical Co., Ltd.) 100μ! , bovine fibrinogen (
Type 2: 3mg/1111.20ml Tr
is-MCI, 0.15M NaCl, pH 7.5: Dai-Kagakuyakuhin Co., Ltd.) 100 μl and 0.9% NaCl, 1
mg/'ml BSA, 0, 54, 108 or 162 nM MTMIO dissolved in 0.1% Lubrol PX
The reaction was started by mixing 100 μm of the solution, and the time until solidification was measured. Measurement is done using automatic blood coagulation meter Crotic I.
I (manufactured by Metek). The results are as follows. The numerical unit is seconds. [0057] [Table 2] [00581] As shown in Table 2, MTMIO suppressed the coagulation activity of thrombin. [0059]

As described above, the recombinant human thrombomodulin derivative of the present invention is a recombinant human thrombomodulin derivative with a novel structure in which the amino acid sequence has been changed so that sulfated glycosaminoglycans are not added, and the DNA sequence has been changed. It has a longer half-life in the blood than conventional recombinant human thrombomodulin made without modification. On the other hand, like conventional recombinant human thrombomodulin, it does not lose its ability to suppress the coagulation activity of thrombin and its ability to promote protein C activation by thrombin. Therefore, the recombinant human thrombomodulin derivative (MTMIO) of the present invention is highly useful as a new anticoagulant.

[Brief explanation of the drawing]

FIG. 1 is a construction diagram of plasmid p7TMO1 containing the human thrombomodulin gene.

[Fig. 2] Plasmid p7TM17 carrying the full-length human thrombomodulin gene with almost all the 5' non-coding region removed.
This is a construction diagram.

FIG. 3 is a construction diagram of plasmid p7TM19 in which a terminator sequence is linked to the full-length human thrombomodulin gene.

[Fig. 4] Plasmid pTMs07 containing a partial-length human dragonfly modulin gene obtained from plasmid p7TM19.
This is a construction diagram.

FIG. 5 is an explanatory diagram of the construction of plasmid pD-gptB-84.

FIG. 6 is an explanatory diagram of the construction of plasmid pTEN-gptB-23.

FIG. 7 is an explanatory diagram of the construction of plasmid pN-gptB-16.

FIG. 8 is an explanatory diagram of the construction of plasmid pB-neo containing the marker gene neo'.

[Figure 9] Human thrombomodulin expression vector pRS7
It is a construction explanatory diagram of TM-neO.

FIG. 10 is an elution pattern diagram of a Q-Sepharose column chromatogram of a culture solution of human thrombomodulin-producing cells.

FIG. 11: Active substance TM of recombinant human thrombomodulin
-α and TM-β elution patterns by reverse phase HPLC under acidic conditions. In the figure, (A) is of TM-α, (
B) shows the elution pattern diagram of TM-β.

[Figure 12] Reversed phase H of TM-β under weak alkaline conditions
It is an elution pattern diagram by PLC. FIG. 13 is an elution pattern diagram of an ion exchange chromatogram of TM-β. FIG. 14 is an HPLC elution pattern diagram of an unsaturated trisaccharide (ΔDi-XS) obtained by treating TM-β with chondroitinase ABC. [Figure 15] ΔDi-45 was mixed with unsaturated trisaccharide (ΔDi-XS) obtained by treating TM-β with chondroitinase ABC. It is an LC elution pattern diagram. FIG. 16 is an HPLC elution pattern diagram of unsaturated trisaccharide obtained by treating TM-β with chondroitinase ACI flavo. FIG. 17 is an HPLC elution pattern diagram of ΔD 1-XS and standard samples treated with Chondro 4-sulfatase.

FIG. 181 is a diagram showing the effect of TM-β on promoting protein C activation of thrombin. FIG. 19: TM-β treated with chondroitinase ABC
FIG. 3 is a diagram showing the half-life of rat blood concentration of untreated TM-β. In the figure, -〇- is for untreated TM-β, -・-
shows the results of chondroitinase ABC-treated TM-β.

[Figure 20] Plasmid in which a partial specific mutation has been introduced into the partial-length human dragonfly modulin gene portion l) M2TMDO7
It is a construction explanatory diagram.

FIG. 21 is an explanatory diagram of the construction of the human thrombomodulin derivative expression vector pR37M2TM-neo.

FIG. 22 is a diagram showing the elution pattern of the recombinant human thrombomodulin derivative MTMIO according to the present invention by reverse phase HPLC under acidic conditions.

FIG. 23 is a diagram showing the rat blood concentration half-life of the recombinant human thrombomodulin derivative MTMIO according to the present invention. It is expressed as the relative residual amount when the blood concentration 3 minutes after administration is taken as 100%.

FIG. 24 is a diagram showing the effect of promoting protein C activation of thrombin by the recombinant human thrombomodulin derivative MTMIO according to the present invention.

[Figure 3]

[Figure 5]

[Figure 9]

[Figure 18]

[Figure 24]

Claims (7)

[Claims] Claim 1: Amino acid 472 of human thrombomodulin
The serine, glycine, serine, glycine, glutamic acid part from position 476 and the continuous amino acid sequence around it are changed by removing, adding, or substituting amino acids, and the sulfated glucosamine is cleaved by chondroitinase ABC. Recombinant human thrombomodulin derivative demodified with Noglycan Claim 2: Consists of an amino terminal region, an EGF-like region, and an O-glycosylation site region among the regions constituting human thrombomodulin, and comprises amino acids 472 to 4.
The continuous amino acid sequence of the 76th serine, glycine, serine, glycine, glutamic acid part and its surroundings is
A recombinant human thrombomodulin derivative that has been modified by removing, adding, or substituting amino acids and is no longer modified with sulfated glycosaminoglycans that are cleaved by chondroitinase ABC. Claim 3: Consists of a region that lacks the transmembrane region and intracytoplasmic region among the regions constituting human thrombomodulin, and comprises the serine, glycine, serine, glycine, and glutamic acid portions from amino acid positions 472 to 476 and their surroundings. A recombinant human thrombomodulin derivative in which the continuous amino acid sequence of is changed by removing, adding, or substituting amino acids and is no longer modified with sulfated glycosaminoglycan that is cleaved by chondroitinase ABC. 4. A region constituting human thrombomodulin that includes the 1st alanine to the 491st alanine from the N-terminus, and the serine/glycine/serine/glycine/glutamic acid moiety from amino acid positions 472 to 476, and A recombinant human thrombomodulin derivative in which the surrounding continuous amino acid sequence is changed by removing, adding, or substituting amino acids and is no longer modified with sulfated glycosaminoglycan that is cleaved by chondroitinase ABC. Claim 5: Plasmid pRS7M2TM-neo [Claim 6]E. coli RS7M2TM-neo Claim 7: Cultured cells transformed with plasmid pRS7M2TM-neo