JPH07119237B2 - Hirudin mutant, its production method and anticoagulant - Google Patents

Description

TECHNICAL FIELD The present invention relates to a hirudin mutant, a method for producing the same, and an anticoagulant containing the hirudin mutant as an active ingredient.

[Problems to be Solved by Prior Art and Invention]

Hirudin, an anticoagulant factor secreted by the salivary glands of the medicinal leech ( Hirudo medicinalis ), is a mixture of peptides consisting of 65 and 66 amino acids. The structure of hirudin was elucidated by Dodt et al. [FEBS Lett. 165 , 180 (1984)], which corresponds to hirudin variant (Variant) 1 (HV1) and is another hirudin variant HV2 [Harvey et al. Proc. Na.
tl.Acad.Sci.USA. 83, 1084 (1986)] have different HV1 and 9 amino acids, which is yet another hirudin variant HV3 [D
odt et al. Biol. Chem. Hoppe-Seyler. 367 , 803 (1986)],
Same as HV2 up to Ser ³² , with amino acid (Ala ⁶³ ) at the C-terminal side
10 amino acids differ, including the addition of. The structures of these three variants are shown in FIG.

These natural variants contain 65 or 66 amino acids,
Two domains can be recognized. That is, a spherical N-terminal portion containing three disulfide bonds and an acidic C-terminal portion showing homology with the thrombin cleavage site or fibrinogen cleavage site in the prothrombin molecule.

The present inventors have found that, in a comparison of C-terminal amino acid sequence of naturally occurring variants hirudin HV1 and HV3, Leu ⁶⁴ -Gln ⁶⁵ of HV1 is HV3
It found that is substituted with Asp ⁶⁵ -Glu ^66. Then, the production of synthetic genes for HV1 and HV3 and their synthesis in E. coli
Patent application (Japanese Patent Application No. 1-207200)
No.)

These hirudin mutants HV1, HV2 and HV3 each have an antithrombin activity, but they cannot be said to be sufficient for use as a clinical drug because they cause prolonged bleeding time as a side effect. The present inventors
Various hirudin mutants were prepared based on the temporary structures of HV1, HV2 and HV3, and their properties were compared in animal models.The amino acid residues 53 and higher of hirudin HV1 were replaced with the amino acid sequences of 53 and higher of HV3. The present inventors have found that the hirudin mutant of HV1 and HV3 (chimeric hirudin) not only has a high antithrombin activity but also reduces the extension of bleeding time, and completed the present invention.

[Means for Solving the Problems]

The present invention relates to a novel hirudin mutant having high antithrombin activity.

The present invention also provides a DNA sequence encoding the amino acid sequence of a hirudin mutant having high anticoagulant activity and reduced bleeding tendency, and a trait obtained by transforming Escherichia coli with an expression vector containing this DNA sequence. The present invention relates to a transformed microorganism and a method for producing a hirudin mutant in which the hirudin mutant is expressed using the transformed microorganism and recovered.

Furthermore, the present invention relates to an anticoagulant containing such a hirudin mutant as an active ingredient.

The novel hirudin variant of the present invention has an amino acid sequence represented by the following primary structural formula.

The DNA sequence encoding the amino acid sequence of this mutant is represented by the following formula, for example.

GTT GTA TAC ACT GAT TGT ACT GAA TCT GGC 30 CAG AAC
CTG TGT CTG TGT GAA GGA TCC AAC 60 GTT TGT GGT CA
G GGT AAC AAA TGT ATC CTC 90 GGG TCT GAT GGT GAA A
AG AAC CAG TGT GTT 120 ACT GGT GAA GGT ACC CCG AAA
CCG CAG TCT 150 CAT AAC CAG GGT GAT TTC GAA CCG A
TC CCG 180 GAA GAC GCG TAC GAT GAA The hirudin variant represented by (Formula I) of the present invention may be produced by chemical synthesis, or may be produced by a genetic engineering technique.

In order to produce it by a genetic engineering method, as shown in Examples described later, first, hirudin HV1 secretion plasmid pMTSHV1 and
pMKSHV1 is constructed, and E. coli is transformed with this, and the transformed microorganism is used to secrete and produce hirudin HV1.
As shown in FIG. 3, this plasmid pMTSHV1 contains a promoter (Ptrp), a DNA sequence encoding a signal peptide (Pho A signal), a DNA sequence encoding the amino acid sequence of hirudin HV1 and a DNA sequence containing a transcription termination signal. (RrnBT ₁ T ₂ ). In the present invention, hirudin
A DNA sequence encoding the amino acid sequence of Hildin HV1 from position 53 onwards by cutting the HV1 secretion plasmid pMTSHV1 with a restriction enzyme in order to replace the amino acid sequence of amino acid residue 53 onwards of HV1 with the amino acid sequence of position 53 onward of HV3. except for. On the other hand, HV3
The DNA sequence encoding the amino acid sequence from the 53rd position of the plasmid pUCHV3 that expresses is excised using a restriction enzyme.
This plasmid pMTSHV1 is reacted with DNA excluding the DNA sequence encoding the amino acid sequence at the 53rd position of hirudin HV1 and the DNA encoding the amino acid sequence at the 53rd position of hirudin HV3 derived from the plasmid pUCHV3 using a DNA ligase or the like. DNA encoding the amino acid sequence of the hirudin variant of the present invention
Construct the plasmid pMTSHV1C3 containing

This plasmid pMTSHV1C3 contains a promoter derived from the plasmid pMTSHV1, a DNA sequence encoding a signal peptide, a DNA sequence encoding the amino acid sequence from the 1st position to the 52nd position of hirudin HV1, a transcription termination signal and a plasmid pUC.
It comprises a DNA sequence encoding the amino acid sequence of Hildin HV3 derived from HV3 after position 53, wherein the DNA sequence is located between the DNA sequence encoding the amino acid sequence of positions 1 to 52 of Hirdin HV1 and the transcription termination signal. It is incorporated.

Escherichia coli is transformed by incorporating this plasmid pMTSHV1C3 into Escherichia coli, and the transformed microorganism is cultured to produce the hirudin mutant of the present invention in the cells and the culture solution.

In the present invention, the hirudin mutant is isolated by a generally known method and purified by a chromatograph, reverse phase HPLC or other purification method.

The obtained hirudin mutant was hirudin in an animal model.
It exhibits a higher antithrombin activity and a lower bleeding tendency than HV1, and can be used as an excellent anticoagulant by preparing a formulation by a method generally known as a method for producing a conventional formulation. That is, the hirudin variant of the present invention is prepared by any conventional method using any conventional pharmaceutical carrier or excipient. Administration can be carried out parenterally, for example intravenously, intracutaneously, subcutaneously or intramuscularly, and topically. The dose is appropriately determined according to each case in consideration of symptoms, age of the recipient, sex, etc., but is usually 0 per adult per day.
It is 1 to 100 mg, which is administered once or several times a day in divided doses.

The plasmid pUCH for constructing the hirudin HV1 secretion plasmids pMTSHV1 and pMKSHV1 used in the present invention.
A method for constructing V1, pMT1 and pMK2 and a method for constructing plasmid pUCHV3 for constructing plasmid pMTSHV1C3 are shown as reference examples.

Reference Example 1 Preparation of plasmid pUCHV1 and plasmid pUCHV3 Using 10 units of pUC18 and 30 units of EcoRI and 30 units of HindIII
Digested at 37 ° C for 2 hours. This was subjected to agarose gel electrophoresis to extract the vector portion, the protein was removed by phenol extraction and the precipitate was precipitated with cold ethanol.
Was dissolved in TE buffer (10 mM Tris-HC1, pH 8.0, 1 mM EDTA). Add 50 ng of this solution to the equivalent of HV1 or HV3 duplex
10 μl containing DNA (66 mM Tris C1, pH 7.5, 5 mM MgCl ₂ ,
5 mM DTT, 1 mM ATP, T4 DNA ligase 300 units)
After overnight reaction at 6 ° C, EcoRI and HindI of plasmid pUC18 were used.
A plasmid pUCHV1 in which the HV1 gene was inserted between II and II, and a plasmid pUCHV3 in which the HV3 gene was inserted were obtained.

Reference Example 2 Preparation of plasmid pMK2 and plasmid pMT1 (a) Preparation of plasmid pMK2 Commercially available plasmid pKK223-3 (Pharmacia) was digested with restriction enzymes PvuI and NruI to obtain a fragment containing the tac promoter and commercially available pUC18. Restriction enzymes PvuI and Pvu
The replication origin (Ori) obtained by cutting with II and a fragment containing the ampicillin resistance gene were ligated with T4 ligase. This was introduced into Escherichia coli JM109 strain, cultured, and screened for ampicillin resistance to obtain a vector having the tac promoter and the replication origin (Ori) of pUC18. This plasmid was designated as pMK2.

(B) Preparation of plasmid pMT1 10 μg of this plasmid pMK2 was added to 30 units of EcoRI and Eco47I.
After digestion with II, the fragment containing the tac promoter was removed, and the fragment containing the replication origin was recovered by agarose gel electrophoresis. On the other hand, the base sequence of the trp promoter was synthesized by a DNA synthesizer. This fragment and the pMK2 were paired with EcoRI and Eco47.
The fragment digested with III was reacted with T4 DNA ligase at 16 ° C. overnight. This was used to transform E. coli JM109 strain and
A vector having an rp promoter and an origin of replication (Ori) of pMK2 was prepared. The nucleotide sequence of this plasmid was confirmed by the method of Sanger etc. and designated as pMT1.

The present invention will be specifically described by showing Examples of the present invention.

Example 1 (1) Preparation of hirudin HV1 secretion plasmid pMTSHV1 The plasmid pMTSHV1 was constructed according to the method shown in FIG. First, E. coli alkaline phosphatase (phoA)
Signal peptide of N-terminal amino acid Val of hirudin HV1
To construct a DNA fragment corresponding to ¹ -Val ^2, it was synthesized four oligonucleotides shown in Figure 2. After deprotection, each oligonucleotide was purified by 10% polyacrylamide gel electrophoresis.

After phosphorylating 500 pmol of each of two kinds of oligonucleotides (S2, S4), 20 pmol of each of four kinds of oligonucleotides were mixed and annealed. 20 μl of a solution containing T ₄ DNA ligase
The reaction was carried out at 16 ° C. overnight. The protein was removed with phenol and chloroform, followed by precipitation with cold ethanol to obtain the desired double-stranded DNA fragment. 1/10 amount of this fragment and restriction enzyme E
pUCHV1 (patent application 1-207200) cut with coRI and AccI 10
0 ng was reacted with T ₄ DNA ligase at 16 ° C. overnight.
Using this, Escherichia coli JM109 strain was transformed to obtain a hybrid plasmid pSHV1 containing a fusion gene in which a DNA fragment encoding hirudin HV1 was linked immediately after the region encoding the phoA signal peptide. The nucleotide sequence of this plasmid pSHV1 was confirmed by the method of Sanger et al.

Use this pSHV1 (10 μg) with 30 units of restriction enzyme EcoRI, HirdIII
It was digested with 30 units and subjected to agarose gel electrophoresis to purify the 275 bp fusion gene fragment.

100 ng of this DNA fragment and E. coli expression vector pMT1 (Japanese Patent Application No.
-212442) was digested with restriction enzymes EcoRI and HindIII, and then purified by agarose gel electrophoresis.
00 ng was reacted with T ₄ DNA ligase at 16 ° C. overnight. Escherichia coli JM109 strain was transformed with this reaction solution, and the phoA signal peptide and hirudin HV1 were placed downstream of the trp promoter.
A hirudin HV1 secretion plasmid pMTSHV1 ligated with a fusion gene coding for p. The nucleotide sequence of this plasmid pMTSHV1 was confirmed by a method such as Sanger.

(2) Preparation of hirudin HV1 secretion plasmid pMKSHV1 The plasmid pMKSHV1 was constructed according to the method shown in FIG.

A fusion gene encoding the above-mentioned phoA signal peptide and hirudin HV1 and the E. coli expression vector pMK2 (Japanese Patent Application No. 1-21
2442) was digested with restriction enzymes EcoRI and HindIII and reacted with T ₄ DNA ligase to transform Escherichia coli JM109 strain to obtain hirudin HV1 secretion plasmid pMKSHV1 in which the fusion gene was ligated to the downstream of tac promoter. The nucleotide sequence of this plasmid pMKSHV1 was confirmed by the method of Sanger, etc.

(3) Secretion production of hirudin by hirudin HV1 secretory plasmid Escherichia coli E. coli JM109 strain transformed with the plasmids pMTSHV1 and pMKSHV1 constructed in (1) and (2) above was used in 2xYT medium containing 100 μg / ml of ampicillin (Bacto. Tryptone 16g / l, Bacto yeast extract 10g / l, N
aCl5g / l). After culturing at 37 ℃ for 24 hours, 1 ml of culture solution
Was collected.

Each sample of the precipitated cells was suspended in 1 ml of 25% sucrose, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA and treated at room temperature for 10 minutes. After collecting the cells by centrifugation at 6000 × g for 10 minutes, the cells were suspended in 1 ml of cold water and subjected to osmotic shock to release the substance in the periplasmic space of the cells. The cells were removed from the periplasmic fraction by centrifugation at 6000 × g for 10 minutes, and the antithrombin activity in the supernatant was measured to measure the amount of hirudin secretory accumulation. Antithrombin activity is a synthetic substrate for thrombin, chromozyme TH (tosylglycylprolyl arginine 4
-Nitroanilide acetate, manufactured by Boehringer Mannheim) The degree of inhibition of hydrolytic activity was measured by a colorimetric test.

The reaction was carried out with 100 mM Tris.HCl (p
H8.5), 150 mM NaCl, 0.1% polyethylene glycol 600
0.36 U of human thrombin (manufactured by Sigma) was added to the buffer consisting of 0, standard hirudin or an unknown sample was added to this thrombin reaction mixture, and pre-incubated at 37 ° C for 3 minutes.

Substrate chromozyme TH was added to a final concentration of 200 μM, and the release of P-nitroanilide was measured at a wavelength of 405 nm.
The increase in absorption per minute was determined and the antithrombin activity (ATU) was measured.

As a result, an antithrombin activity of 450 ATU per ml of the medium was observed in the strain having the plasmid pMTSHV1. In the strain containing the plasmid pMKSHV1, 360 ATU of antithrombin activity was observed per 1 ml of medium. In addition, the plasmid pMTSHV1
Various transformations of E. coli JM101 and JM10 were carried out by transformation methods such as anahan.
Table 1 shows the results of investigation into secretory production after introduction into 3, JM109, TG1, HB101, JA221, IF03301, C600, RR1, DH5.

When RR1 was used as a host, about 2000 ATU / ml of HV1 was secreted and produced.

(4) E. coli JM109 / pMTSHV1 transformant in a 5 l fermenter
Fermentation and secretion of hirudin HV1 into the culture broth in the same manner as described in (3) above, in a 5 l fermenter transformed with the plasmid pMTSHV1 in 2 l of 2xYT medium containing 2% glucose. coli JM109 strain ( E.col
i JM109 / pMTSHV1) was subjected to aeration and stirring culture at 37 ° C for 24 hours, and about 5300 ATU per ml of hirudin was secreted and produced in the culture.

(5) Purification method of hirudin HV1 from the culture medium After the fermentation, 1.5 l of the culture medium was collected and centrifuged (6000 ×
cells for 10 minutes) to separate the cells from the supernatant. When the salt concentration of the supernatant was measured by a salinometer, it was 1.3%, so this was diluted 4 times with 10 mM potassium phosphate buffer (pH 7.0) and filtered through a 3.2 μm filter (manufactured by Pall). . The obtained filtrate was added with 10 mM potassium phosphate buffer (pH 7.
0) Equilibrated with QAE-Toyopearl column (4.4 x 7 cm)
I went to After application, wash with equilibration buffer and then 0.2M NaCl
Hirudin HV1 was eluted stepwise. Concentrate the eluate using Amicon Diaflow membrane (YM5) to obtain 10 mM
Gel filtration was carried out on a column of Sephacryl S-100 equilibrated with a potassium phosphate buffer (pH 7.0).

Active fraction is further added with 10 mM potassium phosphate buffer (pH 7.0)
Was added to a DEAE-Toyopearl column (4.4 x 40 cm) equilibrated with and washed thoroughly, then eluted with a linear gradient of 3 l of equilibration buffer and 3 l of 0.3 M equilibration buffer in sodium chloride. Finally, C ₄ due to the Waters Delta Prep 3000
Purification was carried out on a reverse phase HPLC column. Column is 0.065%
Hirudin was eluted from the column with a linear gradient of (v / v) trifluoroacetic acid and 15-30% (v / v) acetonitrile.

When the amino acid composition and N-terminal amino acid sequence of the purified hirudin HV1 were examined, the amino acid composition value showed the value of natural HV1 as shown in Table 2 and the N-terminal sequence was Val- as shown in Table 3. It was Val-Tyr, and it was found that the phoA signal peptide was correctly cleaved. As a result of measuring the antithrombin activity, it was 9600 ATU / mg.

(6) Preparation of chimeric hirudin HV1C3 secretion plasmid pMTSHV1C3 The plasmid pMTSHV1C3 was constructed according to the method shown in FIG. To replace the amino acid sequence of amino acid residue 53 of hirudin HV1 with the sequence of HV3, HV1 secretion plasmid
pMTSHV1 (10 μg) was digested with KpnI30 units and HindIII30 units of restriction enzymes and subjected to agarose gel electrophoresis,
A 2.8 Kbp DNA fragment was purified.

Similarly, 10 μg of plasmid pUCHV3 (Japanese Patent Application No. 1-207200) was digested with KpnI and HindIII to purify an 80 bp DNA fragment having the C-terminal amino acid sequence of HV3.

100 ng of both DNA fragments were reacted with T ₄ DNA ligase at 16 ° C. overnight, and Escherichia coli JM109 strain was transformed with this reaction solution to obtain chimeric hirudin HV1C3 secretion plasmid pMTSHV1C3. The nucleotide sequence of this plasmid pMTSHV1C3 was confirmed by a method such as Sanger.

(7) Secretory production of chimeric hirudin by chimeric hirudin HV1C3 secretion plasmid E. coli E. coli RR-1 strain transformed with the plasmid pMTSHV1C3 constructed in (6) above ( E.coli RR-1 / pMTS
HV1C3 was deposited with the Institute of Microbial Science and Technology, and Microtechnology Research Institute No. 3130) was cultured in 2xYT medium containing 100 µg / ml ampicillin. After culturing at 37 ° C. for 24 hours, 1 ml of the culture solution was collected, osmotic shock was applied to the cells, and the antithrombin activity of the periplasm fraction was measured.

As a result, 3060 ATU of antithrombin activity was observed per 1 ml of the medium.

(8) E. coli JM109 / pMTSHV1 transformant in a 5 l fermenter
Fermentation of C3 and secretion of chimeric hirudin into culture medium Escherichia coli E. col transformed with plasmid pMTSHV1C3
i JM109 strain ( E.coli JM109 / pMTSHV1C3 deposited at the Institute of Microbial Science and Technology, Microtechnology Research Institute No. 3104) in a 5 l fermenter in 2 x YT medium containing 2 l of 2% glucose 37
After agitating and culturing for 24 hours at ℃, 350 ATU / ml in the periplasm, 5700 ATU / ml in the culture broth, totaling 605
An antithrombin activity of 0 ATU / ml was observed.

(9) Purification of chimeric hirudin Purified from the culture solution obtained in (8) above according to (5) above. When the amino acid composition of the purified chimeric hirudin HV1C3 was examined, it was confirmed that the C-terminal amino acid sequence was changed as shown in Formula 1. Specific activity 11,250AT
It was U / mg. Figure 6 shows the HPLC p of purified HV1 and HV1C3.
showed rofile. This condition is VYDAC C4 (0.37 × 25cm)
Column using 15-30% acetonitrile.
A linear gradient was performed at a speed of l / min for 30 minutes.

Example 2 Inhibitory Effect of Hirudin on Thrombin-Induced Lethal Reaction Male mice (20 to 25 g) were anesthetized with thrombin (10 NIH).
Unit / 10 g) was intravenously injected, and the antithrombin effect of the test compound was evaluated using the disappearance of the righting reflex and lethality as indexes.
All test compounds were dissolved in physiological saline and intravenously injected at a dose of 0.05 ml / 10 g 5 minutes before the administration of thrombin. The results are shown in Table 4.

As is clear from the results, the chimeric hirudin HV1C3 of the present invention
Showed a strong inhibitory effect about 4 to 5 times higher than hirudin HV1 in the thrombin-induced lethal reaction in vivo. The inhibitory effect was similar to that of hirudin HV3.

Example 3 Effect of prolonging bleeding time Using male mice (20 to 25 g), pentobarbital (4
(0 mg / kg, ip) A test compound was administered through the tail vein under anesthesia, and 5 minutes after that, a bleeding time at the wound was created by inserting a 21G injection needle (outer diameter 0.85 mm) into the tail vein on the reaction side of the compound administration. Was measured.

The bleeding time was the time when the wound was wiped with a filter paper every 15 seconds and no blood spots were observed on the filter paper. The results are shown in Table 5.

In general, the side effect of anticoagulants includes the action of extending bleeding time. However, it was confirmed that the chimeric hirudin HV1C3 of the present invention had a significantly lower bleeding tendency than hirudin HV1 and hirudin HV3.

Example 4 Preparation containing chimeric hirudin HV1C3 The purified chimeric hirudin HV1C3 obtained in Example 1 (9) was desalted with Sephadex G25 (Pharmacia) and then sterile filtered with a 0.22 μm filter. This solution was dispensed into a vial and freeze-dried. The lyophilized powder of chimeric hirudin HV1C3 thus obtained can be dissolved in physiological saline to be used as an injection.

〔The invention's effect〕

The hirudin mutant of the present invention has a stronger antithrombin action than hirudin HV1 and has a lower bleeding tendency, and is therefore useful as an anticoagulant.

In addition, the hirudin mutant of the present invention can be produced economically in large quantities because it is produced by a genetic engineering technique.

[Brief description of drawings]

FIG. 1 shows the amino acid sequences of HV1, HV2 and HV3 natural mutants of hirudin, and FIG. 2 can be used in the present invention.
The base sequence of phoA signal bebtide is shown. FIG. 3 shows a conceptual diagram of the construction method of the hirudin HV1 secretion plasmid pMTSHV1, and FIG. 4 shows a conceptual diagram of the construction method of pMKSHV1. FIG. 5 shows a conceptual diagram of a method for constructing a secretion plasmid of the hirudin mutant (HV1C3) of the present invention. FIG. 6 (a) is a reverse phase HPLC chromatogram of purified hirudin HV1, and FIG. 6 (b) is a reverse phase HPL of purified HV1C3 of the present invention.
Chromatograms of C are shown respectively.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 15/09 ZNA C12P 21/02 C 9282-4B // (C12P 21/02 C12R 1:19)

Claims (5)

[Claims] 1. A hirudin mutant having the following amino acid sequence: 2. A DNA sequence encoding a polypeptide having the amino acid sequence of formula (I). 3. A transformed microorganism obtained by transforming Escherichia coli with an expression vector containing a DNA sequence encoding a polypeptide having the amino acid sequence of formula (I). 4. A transformed microorganism according to claim 3 is cultured to express the hirudin mutant represented by the formula (I),
A method for producing the hirudin mutant according to (formula I), which comprises recovering this 5. An anticoagulant comprising the hirudin mutant according to (formula I) as an active ingredient.