JPH01247092A - Production of thrombin inhibitor - Google Patents

Abstract

PURPOSE:To efficiently produce desulfatohirudin protein, by culturing an Escherichia coli transformed with a plasmid into which the DNA coding the hirudin protein of thrombin-inhibiting activity is inserted. CONSTITUTION:A DNA sequence containing the DNA coding the hirudin protein which inhibits the thrombin is inserted into the manifestation vector to form a recombinant plasmid. The recombinant plasmid is used to transform Escherichia coli and the transformed product is cultured. The culture mixture is centrifuged to collect the cell bodies, then, the cell bodies are treated with ultrasonic waves in a buffer solution and lysozyme or dynomill to prepare the supernatant. The supernatant is fractionated using ammonium sulfate or subjected to gel filtration with Sephadex G-50 or the like to obtain the desulfatohirudin protein.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing desulfatohirudin protein, and more specifically, to a method for producing a desulfatohirudin protein, and more specifically, to a method for producing a novel synthetic gene encoding desulfatohirudin IIV-1. The present invention relates to a recombinant plasmid containing the recombinant plasmid, a host transformed with the recombinant plasmid, and a method for producing desulfatohirudin protein using the same.

[Prior art and problems] Hirujin is a leech, Hirudo medicinalis (
It is an anticoagulant present in the former rudo medicinalis. Currently in Hirujin! 1 Sea 1 (F
ebs.

Letters + Tadashi (2) 180-184 (1984)
), HV-2 (Proc, Na-tl, 8cad, Sc
i,,11. s, A, 83 1084 ~ 108B (1
Three types are known: 986)) and des(1/al)zhirudin (European Patent Application No. 158,986). These three types of hirudin differ only in a few amino acids in their structure, with a hydrophobic amino acid at the N-terminus, a polar amino acid at the C-terminus, a tyrosine residue (Tyr”) existing as a sulfate monoester, and three They each have disulfide bridges, anticoagulant activity, etc. In contrast to heparin, which is used for anticoagulant treatment, hirudin does not act through antithrombin■, but directly. Hirudin is unique in that it acts on thrombin. Conventionally, hirudin has been obtained from leech extract, which requires time and expense to separate and purify, and furthermore, it is extremely difficult to obtain raw materials in large quantities, making it impractical. , other manufacturing methods were strongly desired.

[Means for Solving the Problems] Hosts other than leeches lack sulfate transferase, and the hirudin protein obtained by genetic recombination technology is expected to be desulfatohirudin. However, it is already known that desulfatohirudin, which is obtained by chemically treating hirudin produced three times from leech to remove the sulfato group, has a thrombin inhibitory activity comparable to that of leech-derived hirudin (European patent application 142nd,
No. 860). This suggests that it is useful to produce large quantities of desulfatohirudin protein using genetic recombination technology.

Therefore, as a result of intensive research into a method for efficiently producing a protein with hirudin activity, the present inventors found that D.
By synthesizing NA, incorporating it into an expression vector using recombinant technology, and introducing it into E. coli, we succeeded in easily producing a large amount of desulfatohirudin protein.

That is, in chemically and biochemically synthesizing and joining the hirudin HV-1 gene, in addition to the DNA sequence encoding hirudin HV-1, a stop codon such as TAA is added.
was placed on the 3'' side, and for example, EcoRI and Bam1lI cohesive terminal DNA sequences were attached to the 5 and 3 ends, respectively, for insertion into the hefter.

The codons shown in Figure 1 were selected and divided into 7 fragments. Figure 2 shows the t)NA of each DNA fragment.
Here is an example of an array. As long as care is taken to avoid self-association, the method of dividing the fragments is not limited to that shown in FIG. 1, and various methods are possible. Each DNA
Fragments may be synthesized according to known synthetic methods. Each D
The 5' ends of the NA fragments are phosphorylated if necessary, two or three of them are mixed and hybridized, and then double-stranded DNA is made with DNA ligase. Alternatively, all the trees can be mixed together and hybridized, D
It is also possible to make double-stranded DNA using NA ligase (Figure 3). This was ligated with a vector obtained by digesting pBR322 with EcoRI and BamHT to obtain a new plasmid p3010 (Fig. 4). As a result of isolating this plasmid DNA and examining the DNA sequence, the presence of the target hirudin HV-1 gene was confirmed.

The obtained plasmid can be easily transferred to Escherichia coli, such as MC1061, JM107, HBIOI,
It can be transformed into microorganisms such as DE5α.

Examples of expression plasmids include P and L.

p223-3, pYEJOOl, pD, which are listed in the catalogs of Chemical Co., Pharmacia Co., etc. and are commercially available.
R540, pDR720, pNEo, etc. can be used. For example, convert plasmid p3010 DNA into Ec
After digestion with oRI and Nrul, the EcoRI-Nrul DNA fragment was separated and recovered on polyacrylamide gel and the expression plasmid pKK223-3 was transformed into Eco
It was inserted into a vector obtained by digestion with RI and Smal to obtain expression plasmid p4014 (Fig. 5). Strain MTE4014 was transformed into MC1061.
(FURM P-9924) was obtained. In addition, strains transformed into Escherichia coli JM107, HBIOI, and DE5α were also obtained in the same manner.

These strains can be easily cultured under the same conditions as the host strain, except that they have acquired ampicillin resistance and express and produce desulfatohirudin protein. The target protein accumulated in the bacterial cells can be separated and recovered by a conventional protein recovery method after the bacterial cells are lysed. For example, after culturing, the bacterial cells are collected using a centrifuge, and treated with ultrasound, lysozyme, or Dynomil in a buffer solution to obtain a supernatant, and this supernatant is subjected to ammonium sulfate fractionation or Sephadex G- The target protein can be obtained by gel filtration such as 50.

The present inventors have confirmed that the recombinant E. coli cell extract of the present invention has extremely strong thrombin inhibitory activity, and have purified and identified the desired desulfatohirudin protein from this extract, thereby completing the present invention. reached.

〔Example〕

Next, the present invention will be explained by examples, but the present invention is not limited to these examples in any way.

Example 1 Chemical synthesis of DNA fragments and their purification (Fig. 2) Seven DNA fragments were each synthesized using a fully automatic synthesizer (manufactured by Applied Systems) by the phosphoramidite method. A commercially available product (manufactured by Systik) was used as a protected monomer serving as a raw material. After the synthesis was completed, the reaction product was transferred to a container, 1.5 m of concentrated ammonia water was added, the container was tightly stoppered, and the reaction was allowed to proceed at room temperature for 1 hour and then at 55° C. overnight. After concentration to dryness, 1 ml of 80% aqueous acetic acid was added, and the mixture was allowed to stand at room temperature for 30 minutes, and then concentrated to dryness. The contents were dissolved in water, dialyzed overnight, concentrated, and subjected to electrophoresis on a 20% polyacrylamide gel containing urea, and the desired band portion of the gel was cut out.
1.5 d of NA recovery solution (0.5 M sodium acetate, 10
mM magnesium acetate, 0.1% v/v sodium dodecyl sulfate (SO5), 1 mM disodium ethylenediaminetetraacetic acid (EDTA) (ρ117.5)) were added and the mixture was shaken overnight. Furthermore, ON8 was collected using a short column of cellulose DE-52 (manufactured by Whatman), extracted with phenol-chloroform, and precipitated with ethanol to obtain pure water or NA fragments. The INA fragment synthesized and purified in this manner is shown in FIG.

Example 2 Phosphorylation and fragment ligation reaction (Fig. 3) 1 μg each of DN^ fragments 1.2.3.4.5.6.7 were combined, and 7 units of T4 polynucleotide kinase (manufactured by Takara Shuzo), gamma- 40 pl of phosphorylation solution containing 2 μCi of ATP [500 mM Tris-CI'
(pH 17°5), 100mM MgCIz, 5
mM dithiothreitol (DTT), 0.1mM ED
TA, 0.1mM spermidine] was added and reacted at 37°C for 1 hour. Additionally, 10mM ATP? containing 7 units of T4 polynucleotide kinase? 10μ2 of the 8 solution was added and reacted for 1 hour.

After heating at 95°C for 3 minutes, the mixture was slowly cooled to room temperature and concentrated to dryness. Add 50tIN (66mM Tris) of this solution containing 15 units of T4 ligase (manufactured by Takara Shuzo)
-CI (pH 7,5), 5II+M MgCl! , 5
Add mM DTT, 1mM ATP) and heat at 6°C.
The mixture was allowed to react overnight.

Add 50μl of phenol and 50μl of chloroform,
After extraction, add ethanol and incubate at -80"C for 1 hour at -20
'C for 30 minutes, then centrifuge to obtain the desired double strand D.
NA was collected.

Example 3 Cloning of synthetic gene (Fig. 4) The cloning vector was E. coli plasmid pBR3.
22 was used. [1coRI 4 units, Raw old 15
pBR32210 containing unit, c+ g solution? (150t
t l CIIIM Tris-CI (pH 8), 0
．． After reacting 6GsM MgCh, 0.6+aM mercaptoethanol, 6II+M NaCl) at 37°C for 3 hours, dephosphorylating enzyme (manufactured by CIP Boehringa) was added.
j volume containing 4 units, 12u l (500mM Tr
is-CI (pH 9,0), 1mM MgCIz
, 1mM ZnCI, ) were added and reacted at 37°C for 1 hour. After phenol extraction, it was precipitated with ethanol. Dilution buffer (10mM T
ris-CI, 1mM [1DTA] was added, and 10μl (66mM Tri
s-C1 (pH 7,6), 5mM MgCh,
5mM ATP, 5 units of ligase] was added, and the mixture was allowed to react at 6°C overnight. E. coli using this reaction solution! 'IC1
061 strain was transformed to obtain ampicillin-resistant transformants. Plasmid DNA was prepared from this transformant, the degradation pattern with restriction enzymes was examined, and DNA sequencing was performed.
It was confirmed that the plasmid p3010 had the hirudin IIV-1 synthetic gene inserted between RI and BamHI.

Example 4 Construction of expression plasmid (Figure 5) Expression plasmid pKK223-3 (manufactured by Pharmacia) 10. 4 units of EcoRI and reaction solution (50
mM Tris・CI (pH 7,5), 100mM
NaCl, 10mM MgCh, 1mM DTT]
was added at 1.37"C for 3 hours at 50 .mu.i.

After phenol extraction, cold ethanol precipitation was performed. Sma I buffer (20mM MCI) was added to the residue obtained by centrifugation.
, 10mM Tris-CI (pH 8,0), 10
Add 1g of mM MgC) and 15 units of Sma to 50 μ2
The mixture was reacted at 37°C for one night. After phenol extraction, cold ethanol precipitation was performed.

The residue obtained by centrifugation was treated with a dephosphorylating enzyme in the same manner as described in Example 3, extracted with phenol, and precipitated with cold ethanol.

Dilution buffer (10mM
Expression vector DNA was prepared by dissolving it in Tr-4s-CI, 1mM EDTA).

On the other hand, plasmid P3010 DIJA obtained in Example 3
15 units of EcoRI and Nrul in an amount equivalent to 100 μg
24 units and reaction solution (50mM Tris-CI
(p)17. j) , loOmM NaCl, 10mM
MgCh, 1mM DT? ) was added to give a total pressure of 150 μN, and the reaction was allowed to proceed overnight at 37°C. The gel was subjected to urea-free 5% polyacrylamide gel electrophoresis, and the gel of the desired band was cut out and treated in the same manner as in Example 1 to obtain an EcoR1/Nrul fragment containing the synthetic hirudin gene. The amount was calculated assuming a recovery rate of 80% and dissolved in dilution buffer.

An amount equivalent to 50 ng of vector DNA was combined with an amount equivalent to 1100 n of DNA fragments, and after being treated with ligase in the same manner as in Example 3, E. coli strain MC1061 was transformed.
Strain MTE4014 (FER) with antithrombin activity
MP-9924) was obtained.

Example 5 A transformant (MTE4014J) was obtained by performing the same reaction using E. coli strain JM107 (manufactured by Hesesda Research) in place of the E. coli MCl061 strain described in Example 4.

Example 6 A transformant (?ITE40L411) was obtained by performing the same reaction using E. coli strain 11BIOI (manufactured by Takara Shuzo Co., Ltd.) in place of the E. coli strain MC1061 described in Example 4.

Example 7 Using E. coli DE5α strain (manufactured by Bethesda Research) in place of the E. coli MC1061 strain described in Example 4, the same reaction was carried out to obtain a transformant (MTE4014(1)).

Example 8 L-broth (1% sodium chloride, 0.5% yeast extract, 1% tryptone water?8?ff1) 5d with MTE4
014 strain was planted and cultured at 37°C overnight. 1.5 d of this culture solution was centrifuged to collect bacteria, and then the upper layer was removed. A solution containing 2μ of lysozyme (10mM
Tris-CI, 1mM EDTA aqueous solution) 10
0. ffi was added, heated at 37°C for 30 minutes, and centrifuged. The obtained upper layer was separated. This upper layer solution is 50μ! and 0
．． 25 υ/Id thrombin solution [Mochida Pharmaceutical's bovine-derived lyophilized preparation in 50 d phosphate buffer (pH 7,5) containing 0.5% bovine serum albumin] 50 t
After heating at 37°C for 2 minutes, add fibrinogen solution (Miles
75% C1ottable bovine fibrinogen manufactured by Co., Ltd. was dissolved in 50mM phosphate buffer (pH 7°5) and adjusted to 10μ/Id) 50μ! was added to start the reaction. Antithrombin activity was then measured using Hematotracer ■ (manufactured by Sanko Bioscience). As a result, the production amount of desulfatohirudin protein was 1.5X.
It was 103 ATtl 100 (antithrombin unit/optical density).

[Brief explanation of the drawing]

The drawings are as follows. Figure 1 shows the hirudin HV-1 gene broken down into seven fragments. Figure 2 shows the DNA sequence of the seven synthesized fragments. Figure 3 shows the hirudin I (V-1 synthesized Figure 4 shows the sequential reaction of fragments to obtain the gene, Figure 4 shows the cloning of the synthetic gene, and Figure 5 shows the cloning of the synthetic gene.
The figure shows the construction of an expression plasmid. Patent applicant Mitsui Toatsu Chemical Co., Ltd. Figure 3 Phosphorylation and fragment ligation reaction ↓
Phosphorylation Figure 2 7 GATCCTTATTATTGTA＾8TAT
TCTTCAGG 88TT to CGGACAAAATT TGTTTATGTGAΔGGATCTAATGT
70merCTTC8
3
9 4th circle Cloning of synthetic gene εcoRI Figure 5 Construction of expression plasmid

Claims (1)

[Scope of Claims] 1) A DNA sequence containing DNA encoding a hirudin protein having thrombin inhibitory activity. [There is a gene sequence. 2) A recombinant plasmid obtained by inserting the DNA according to claim 1 into an expression vector. 3) The recombinant plasmid according to claim 2, wherein the expression vector is PKK223-3. 4) E. coli transformed with the recombinant plasmid according to claim 2. 5) A method for producing desulfatohirudin protein, which comprises using the Escherichia coli according to claim 4.