JPH0354555B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention is directed to angiotensin (aspartic acid (AsP) - arginine (Arg) - valine (Val) - tyrosine (Tyr) - isoleucine (Ile)), which has a strong blood pressure increasing effect and is an interesting physiologically active peptide. - A new recombinant plasmid pANG2 that enables mass production of a fusion protein containing a peptide consisting of the eight amino acid sequence of histidine (His) - proline (Pro) - phenylalanine (Phe), hereinafter abbreviated as ANG2. -11, E. coli containing pANG2-11, dihydrofolate reductase-ANG2 fusion protein with ANG2 on the carboxy-terminal side of the enzyme (hereinafter abbreviated as DHFR-ANG2), DHFR
-This invention relates to a method for separating and purifying ANG2 and a method for producing ANG2. The novel recombinant plasmid pANG2-11 of the present invention is shown in FIG.
It has a DNA sequence. The present invention is suitable for fields such as fermentation industry and pharmaceutical industry. Prior Art ANG2 is a blood peptide and a central substance of the renin-angiotensin system, which is thought to control blood pressure. ANG2 is an interesting physiologically active peptide that acts on ANG2 receptors in peripheral arteries, increases blood pressure through vasoconstriction, and acts on the globus layer of the parasympathetic cortex to promote aldesterone secretion. The technical background of the present invention is so-called genetic manipulation technology. However, regarding the efficient production method of ANG2 using genetic manipulation,
unknown. On the other hand, the present inventors have already developed a plasmid vector pTP70-1 for heterologous gene expression (Japanese Patent Application Laid-Open No.
63-46193) and a method for creating a fusion gene using the same (Japanese Patent Application Laid-Open No. 144992/1999). When these methods are used, the amount of the fusion protein obtained as a result of the expression of the fusion gene accumulated in E. coli cells is expected to be about 20% of the total bacterial protein. However, there is no example of using the above expression vector for the production of ANG2. Purpose of the Invention The purpose of the present invention is to develop a method for mass production of ANG2 using genetic engineering techniques, which has not yet been established. The present inventors have already constructed (1) an expression plasmid that expresses a large amount of E. coli DHFR (Japanese Patent Application Laid-open No. 62-69990), and (2) the carboxy-terminal sequence of E. coli DHFR. (3) The plasmid vector pTP70-1 has been constructed, which makes it possible to fuse a heterologous peptide to the carboxy-terminal side of DHFR of E. coli (JP-A-63-63-1). Publication No. 46193), (4)
It has been revealed that the modified DHFR on pTP70-1 is efficiently expressed in E. coli. Utilizing the above knowledge and as a result of intensive research, the present inventors chemically synthesized the ANG2 gene and pTP70-
1, the ANG2 gene and
By creating a fusion gene of the DHFR gene and expressing the fusion gene in E. coli, DHFR-
They discovered that ANG2 can be produced in large quantities, and further revealed that ANG2 can be effectively produced by using DHFR-ANG2, thereby completing the present invention. Structure of the Invention The present invention provides (1) a novel recombinant plasmid pANG2-11 that enables large-scale expression of DHFR-ANG2;
E. coli cells containing pANG2-11, (3) pANG2
-DHFR produced by E. coli containing -11-
ANG2, (4) from E. coli containing pANG2-11.
Separation and purification of DHFR-ANG2, and (5) DHFR-
The invention consists of a method for producing ANG2 using ANG2. (1) Novel recombinant plasmid pANG2-11 Figure 1 shows the entire base sequence of pANG2-11 of the present invention. The figure shows that only one DNA strand sequence of the double-stranded circular DNA is extracted from Hind of the two restriction enzyme Cla sites present in the plasmid.
Cutting recognition site closer to the site, 5′−
The first "A" of ATCGAT-3' is counted as number 1 and is written in the direction from the 5' end to the 3' end. pANG2-11 of the present invention is a novel recombinant plasmid. pANG2-11 has a size of 4651 base pairs and can confer trimethoprim and ampicillin resistance to the host E. coli. pANG2-11 is pTP70-1
It has a structure in which 43 base pairs of chemically synthesized DNA containing a sequence encoding ANG2 is bound to the BamH site. In Figure 1, 533rd to 575th
The sequence up to No. 1 is a sequence derived from chemically synthesized DNA. The other sequences are derived from pTP70-1. Restriction enzymes are used to sequence chemically synthesized DNA.
The Kpn recognition cleavage site, 5'-GGTACC-3' (sequence from position 570 to position 575 in Figure 1), is included. The part derived from pTP70-1 contains
Kpn site is absent. Therefore, pANG2−
11 BamH sites (532nd to 537th in Figure 1)
By replacing the sequence from the sequence up to the Kpn site with another sequence, it is possible to introduce a heterologous DNA in a fixed direction and more easily create a fusion gene with the DHFR gene. The array from 57th to 566th in Figure 1 is as follows:
DHFR-ANG2, in which ANG2 is bound to the carboxy-terminal side of DHFR via methionine (Met)
is encrypted. Upstream of the sequence encoding DHFR-ANG2, there is a sequence that allows efficient expression of the DHFR-ANG2 gene (Japanese Unexamined Patent Publication No. 46193/1983). In other words, the array from 43rd to 50th is
For efficient translation, it is called SD array.
Furthermore, positions 4609 to 4637 are the consensus transcription promoter and contribute to efficient transcription. From this, pANG2−11 is
When introduced into E. coli, a wide variety of DHFR−
Make ANG2. The produced DHFR-ANG2 is soluble in the bacterial body and contains about 20% of the bacterial protein.
Accumulates about %. By this, pANG2
E. coli containing -11 becomes trimethoprim resistant. In addition, pANG2−11 is
It has an ampicillin resistance gene derived from pPT70-1. From this, E. coli into which pANG2-11 has been introduced also exhibits resistance to ampicillin. pANG2-11 is introduced into E. coli and kept in a stable state, and the E. coli containing pANG2-11 has been deposited at the FIKEN as FERMBP-1815. pANG2-11 having such features can be constructed according to Example 1, but the present invention is not limited by the method of constructing the recombinant plasmid. (2) E. coli containing pANG2-11 E. coli containing pANG2-11 shows resistance to trimethoprim and ampicillin.
E. coli containing pANG2-11 is DHFR-
As a result of efficient expression of the ANG2 gene,
DHFR-ANG2 accumulates in large quantities in a soluble state within the bacterial body. Escherichia coli containing pANG2-11 was grown in YT+Ap medium (in 1 liter of medium, 5 g of NaCl,
When cultured at 37° C. until the stationary phase using a liquid medium containing 8 g of tryptone, 5 g of yeast extract, and 50 mg of ampicillin sodium, the accumulated DHFR-ANG2 reaches about 20% of the bacterial protein. When cultured bacterial cells are suspended in an appropriate buffer such as phosphate buffer, disrupted by French press or sonication, and separated into supernatant and precipitate by centrifugation, almost all of the DHFR- ANG2 is recovered in the supernatant. Escherichia coli containing pANG2-11 has been deposited with the Institute of Fine Technology as FERMBP-1815. (3) DHFR-ANG2 Figure 2 shows the DNA sequence encoding DHFR-ANG2 and the amino acid sequence of the protein predicted to be made from it.
DHFR-ANG2 is a novel protein consisting of 170 amino acids. The sequence from positions 1 to 159, counting from the amino terminal side, is a sequence in which one amino acid substitution has occurred in E. coli wild type DHFR (Cys-152 (Wild type) → Glu-152), and from position 163 to Glu-152. The sequence up to the 170th is ANG2. The amino acid immediately preceding the ANG2 sequence is methionine (Met). Due to this,
By treating DHFR-ANG2 with bromcyan, ANG2 can be specifically excised. The isoleucine (Ile)-leucine (Leu) sequences at positions 160 and 161 are BamH of pTP70-1.
This sequence was created to align the reading frame of the genetic code when introducing DNA encoding ANG2 into the site. DHFR produced by pTP70-1
consists of 162 amino acids and is shown in Figure 2.
Of the amino acid sequence of DHFR-ANG2, two amino acid sequences of Gln-Ile are bonded to the sequence from 1st to 160th, counting from the amino terminal side. The molecular weight of DHFR-ANG2 is 19,410. DHFR-ANG2 is a novel protein. Although DHFR-ANG2 has a structure in which ANG2 is fused to the carboxy terminal side of DHFR, it has DHFR enzyme activity. For this reason, when E. coli produces large amounts of DHFR-ANG2, it becomes resistant to trimethoprim, a DHFR inhibitor and antibacterial agent. (4) Separation and purification of DHFR-ANG2 The method for separating and purifying DHFR-ANG2 of the present invention is as follows:
It consists of the following steps: culture of bacterial cells, disruption of bacterial cells, DEAE-Toyopearl column treatment, mesotrixate (MTX)-conjugated affinity chromatography, and DEAE-Toyopearl column chromatography. Cultivation of bacterial cells Culture of E. coli containing pANG2-11 is as follows:
YT + Ap medium (5 g of NaCl in 1 liter of medium,
Liquid medium containing 8 g tryptone, 5 g yeast extract and 50 mg ampicillin sodium. ) can be cultured. Other media include ST+Ap medium (liquid medium containing 2g of glucose, 1g of dipotassium phosphate, 5g of polypeptone, 5g of yeast extract, and 50mg of ampicillin sodium in 1L of medium), etc. Any growth medium can be used, but as far as we have investigated, DHFR-
YT+Ap medium was optimal for ANG2 production. E. coli containing pANG2-11 is inoculated into a medium and cultured at 37°C until the late logarithmic growth phase or stationary phase. Depending on the culture temperature,
The amount of DHFR-ANG2 accumulated varied, and as far as we investigated, the higher the culture temperature, the greater the amount accumulated. The cultured bacterial cells are collected by centrifugation at 5000 rpm. A wet weight of 2 to 4 g of bacterial cells can be obtained from 1 liter of culture medium. Bacterial collection and subsequent operations are performed at low temperatures (between 0 and 10°C, preferably 4°C) unless otherwise specified. Disruption of bacterial cells The bacterial cells obtained by culturing were suspended in buffer solution 1 (10 mM potassium phosphate buffer containing 0.1 mM sodium ethylenediaminetetraacetate (EDTA), PH 7.0) at 3 times the wet weight, Crush the bacterial cells using a French press. Centrifuge the cell suspension at 5000 rpm for 10 minutes to obtain a supernatant. Furthermore, the supernatant is subjected to ultracentrifugation at 35,000 rpm for 1 hour to obtain a supernatant (cell-free extract). DEAE-Toyopearl column treatment This operation is performed for the purpose of pretreatment for the next purification process. Prepare cell-free extract at 0.1M in advance.
Apply to a DEAE Toyopearl column equilibrated with Buffer 1 containing 0.1M KCl, and wash the column with Buffer 1 containing 0.1M KCl. Enzyme elution is
Perform using buffer 1 containing 0.3M KCl.
Fractionate a certain amount of the eluate using a fraction collector. About the fractionated eluate
Measure DHFR activity and collect fractions containing enzyme activity. MTX-bound affinity chromatography The enzyme solution obtained by the above procedure was pre-equilibrated with buffer 1 and then MTX-bound
Adsorb onto Sepharose affinity column. After adsorption, buffer 2 (0.1
Wash with 10 mM potassium phosphate buffer containing mM EDTA, pH 8.5). For washing, measure the absorbance of the eluate from the column at 280 nm, and continue to flow the same buffer until the absorbance becomes 0.1 or less. Elution of the enzyme is performed using Buffer 2 containing 1M KCl and 3mM folic acid, and the eluate is fractionated in fixed amounts using a fraction collector. DHFR for fractionated eluate
Measure the activity and collect fractions containing enzyme activity. The obtained enzyme solution is dialyzed against buffer solution 1 three times. At this stage, DHFR-ANG2 with a purity of 90% or more is obtained. DEAE-Toyopearl column chromatography The dialyzed enzyme solution is adsorbed onto a DEAE-Toyopearl column equilibrated with buffer solution 1 in advance. After adsorption, buffer 1 containing 0.1M KCl
wash with Washing is done by washing the eluate from the column.
Measure the absorbance at 280 nm, and continue to flow the same buffer until the absorbance becomes 0.01 or less. Enzyme elution was performed using buffer 1 from 0.1M to 0.3M.
A linear concentration gradient of KCl is used, and a fixed amount of the eluate is fractionated using a fraction collector. 280nm for fractionated eluate
The absorbance and DHFR activity are measured. Collect fractions with a constant value of enzyme activity/absorbance at 280 nm. Through the above operations, DHFR-ANG2 can be highly purified and homogenized with good reproducibility. According to the present invention, the purification of DHFR-ANG2 consists of
It can be carried out within one week including culturing, and a homogeneous enzyme preparation can be obtained with a recovery rate of 45% or more. DHFR enzyme activity was determined using the reaction solution (0.05mM dihydrofolate, 0.06mM NADPH, 12mM 2-
The measurement is carried out by taking 1 ml of mercaptoethanol and 50 mM phosphate buffer (PH7.0) into a cuvette, adding the enzyme solution to the cuvette, and measuring the change in absorbance at 340 nm over time. One unit of enzyme is
Under the above reaction conditions, it is defined as the amount of enzyme required to reduce 1 micromole of dihydrofolate per minute. This measurement can be easily performed using a spectrophotometer. (5) Production of ANG2 using DHFR-ANG2 Cleavage and separation of ANG2 from purified DHFR-ANG2 is performed by treatment with bromcyan. Lyophilize purified DHFR-ANG2,
Add to this 1 to 10 mg protein/ml.
After adding and dissolving 70% formic acid, add about 20 times the amount of crystalline bromic cyanide to the amount of protein, seal tightly, and react for 24 hours with stirring at room temperature under a nitrogen atmosphere. After diluting the reaction solution with 10 times the volume of water, freeze-dry to remove excess reagents, etc. 1 lyophilized sample
Dissolve in 30% acetic acid to give 10 mg protein/ml. The dissolved sample was collected using an HPLC device (Shimadzu).
LC-4A, inertsil-ODS column)
Elution and separation can be performed using a concentration gradient of 15% to 50% acetonitrile in 0.1% trifluoroacetic acid. Eluates can be detected by measuring absorbance at 220 nm. Figure 3 shows DHFR-
High performance liquid chromatogram of ANG2 sample is shown. The peak approximately 17 minutes after sample injection is ANG2. Separate this peak fraction. After drying the separated eluate using an evaporator, ANG2 can be obtained by adding a small amount of water and freeze-drying to remove the solvent. Furthermore, the amino acid composition can be confirmed by acid hydrolyzing the obtained peptide and then performing amino acid analysis. In the example of the present invention, bacterial cells with a wet weight of approximately 6 g were obtained from 3 liters of culture medium, and this bacterial cell (calculated approximately 48 mg DHFR-ANG2, approximately 2.6 mg
Contains ANG2. ), approximately 23 mg of DHFR−
ANG2 (yield, 48.3%, calculated approximately 1.2 mg of ANG2
including. ), of which 10 mg of DHFR-ANG2 is treated with bromcyane and then separated and purified by HPLC to obtain approximately
We were able to obtain 0.28 mg of ANG2. Next, examples and reference examples of the present invention will be shown. Example 1 Creation of pANG2-11 The DNA encoding ANG2 is 1.5'- GATCCTGATGGATCGCGTGTACATCCA
CCCGTTCTAAGGTACC-3'2.5'- CATCGGTACCTTAGAACGGGTGGATGT
DNA consisting of two 43 nucleotides of ACACGCGATCCATCAG-3' was chemically synthesized according to the phosphoramidite method, and after purification, the 5' end of each DNA was phosphorylated using polynucleotide kinase. Approximately 0.1ml of phosphorylated DNA
(contains about 0.01 μg of DNA) and incubating it at 60°C.
The DNA was annealed (this is called DNA1). Approximately 1 μg of pTP70-1 was cleaved with BamH and then treated with alkaline phosphatase. The alkaline phosphatase-treated DNA was treated with phenol to denature and remove coexisting enzyme proteins, and then the DNA was precipitated with ethanol. After washing the precipitated DNA with 70% ethanol,
The ethanol was removed and the precipitate was dried under reduced pressure.
The operations of DNA cleavage with BamH, alkaline phosphatase treatment, phenol treatment, and ethanol precipitation are all performed using the “Molecular
Cloning A Loboratory Manual” (T.
Maniatis, EFFritsch, J. Sambrook, eds.Cold
Spring Harbor Laboratory (1982), hereinafter referred to as Document 1. ).
The dried DNA was mixed with 50μ of ligase reaction solution (10mM Tris-HCl, PH7.4, _5mM MgCl2,
After dissolving in 10mM dithiothreitol, 5mM ATP), 5μ of DNA1 was added, 1 unit of T4-DNA ligase was added thereto, and the DNA ligation reaction was carried out at 10°C for 12 hours. This reactant,
It was introduced into E. coli according to the transformation method (described in Document 1 above). The treated bacterial cells were transferred to a nutrient agar medium containing 50 mg of ampicillin sodium and 10 mg of trimethoprim (2 g of glucose per 1 liter of the medium,
Contains 1g dipotassium phosphate, 5g yeast extract, 5g polypeptone, and 15g agar. )
By coating on the top and culturing at 37℃ for 24 hours,
We were able to obtain 45 colonies. Select eight colonies from these colonies and incubate them in 1.5 ml of YT+Ap medium (1 liter of medium contains 5 g of NaCl, 5 g of yeast extract, 8 g of tryptone, and 50 mg of ampicillin sodium) at 37°C overnight. The bacterial cells were cultured. Each culture solution was placed in an Etzpendorf centrifuge tube, centrifuged at 12,000 rpm for 10 minutes, and the bacterial cells were collected as a precipitate. Add 0.1ml of electrophoresis sample preparation solution (0.0625M Tris-HCl, PH
6.8, 2% Sodium Lauryl Sulfate (SDS), 10
% glycerin, 5% 2-mercaptoethanol, and 0.001% bromophenol blue. )
was added to suspend the bacterial cells, and this was kept in boiling water for 5 minutes to dissolve the bacterial cells. This treated sample was subjected to SDS-polyacrylamide gel electrophoresis (UK Lammli; Nature, vol. 227, p. 680 (1970)).
Analyzed according to. pTP70 as standard sample
E. coli containing -1 was treated in the same way,
and molecular weight markers such as lactalbumin (molecular weight 14,200), trypsin inhibitor (molecular weight 20,100), trypsinogen (molecular weight 24,000), carbonic anhydrase (molecular weight 29,000), glyceraldehyde 3-phosphate dehydrogenase (molecular weight 36,000), ovalbumin (molecular weight 45,000) and bovine serum albumin (molecular weight 66,000) were run on a 10 to 20% polyacrylamide gradient gel. As a result, in three of the eight colonies, the DHFR band of pTP70-1 disappeared, and a new protein with a clearly larger molecular weight (estimated to have a molecular weight of approximately 22,500) was produced. In addition, the remaining five proteins produce proteins with approximately the same size as DHFR, and the DHFR of pTP70-1 (molecular weight 18379)
was found to migrate as a protein with a molecular weight of approximately 21,000 under these conditions. Select one strain from among the colonies that produce a new protein with a large molecular weight, culture each strain in YT+Ap medium, and use Tanaka and Weisblum's method (T.
Tanaka, B. Weisblum J. Bacteriology,
vol. 121, p. 354 (1975)). The obtained plasmid was named pANG2-11. pANG2-11 is BamH of pTP70-1
It should have a structure in which a chemically synthesized DNA sequence is inserted into the site. The synthetic DNA contains a sequence recognized by the restriction enzyme Kpn, 5′-
GGTACC−3′, is included, so in Kpn
When we tried to cleave pANG2-11, it was indeed cleaved. In addition, EcoR of pANG2-11 (sequences 471-476 in Figure 1) and Sal (sequences 868-476 in Figure 1)
873rd sequence)
For DNA with a length of 400 nucleotides, the dideoxy method using M13 phage (J. Messing; Mehtods
In Enzymology, vol. 101, p. 20 (1983)), the base sequence was determined. As a result, the sequence from position 471 to approximately position 873 of the sequence shown in FIG. 1 was confirmed. The nucleotide sequence of pTP70-1 has been revealed by the present inventors (Japanese Unexamined Patent Publication No. 63-46193). The EcoR-Sal sequence of pANG2-11 is
BamH between EcoR-Sal of pTP70-1
This is a sequence with a length of 43 nucleotides inserted at the site. In addition, the approximately 4.2 kilobase pair DNA obtained by EcoR-Sal cleavage of pANG2-11 is Pst,
The results of restriction enzyme cleavage experiments using Hind, Hpa, Aat, Pvu, Bgl, and Cla revealed that the DNA is exactly the same as the approximately 4.2 kilobase pair DNA obtained by EcoR-Sal cleavage of pTP70-1. Shown. From the above results, it was determined that the entire base sequence of pANG2-11 was the sequence shown in FIG. Example 2 DHFR- produced by E. coli containing pANG2-11
DHFR− produced by E. coli containing ANG2 pANG2−11
The amino acid sequence of ANG2 can be predicted from the base sequence of the DHFR-ANG2 gene. Figure 1
Since the sequence from positions 57 to 566 encodes DHFR-ANG2, the amino acid sequence was estimated using the Torplett code table. As a result, the amino acid sequence shown in FIG. 2 was obtained. From E. coli containing pANG2-11, DHFR-
We isolated and purified ANG2 and investigated the properties of the purified protein. Purification of DHFR-ANG2 A Amount of bacterial cells used: wet weight 6g B Enzyme purification table The purification process in the table is cell-free extract,
DEAE-Toyopearl column treatment, mesotrixate-coupled affinity chromatography, and DEAE-Toyopearl column chromatography.

[Table] DHFR enzyme activity was measured using the reaction solution (0.05mM dihydrofolic acid, 0.06mM NADPH, 12mM 2-mercaptoethanol, 50mM phosphate buffer (PH).
7.0)) was carried out by taking a 1 ml cuvette, adding the enzyme solution thereto, and measuring the change in absorbance at 340 nm over time. One unit of enzyme was defined as the amount of enzyme required to reduce 1 micromole of dihydrofolate per minute under the above reaction conditions. When the obtained enzyme protein was analyzed by SDS electrophoresis (method described in the above example),
Approximately 22,500 single protein bands were shown, indicating that the obtained enzyme preparation was homogeneous. Properties of isolated and purified DHFR-ANG2 When the purified protein exhibiting DHFR activity was examined by enzyme immunoassay,
It was shown to react with antibodies against ANG2.
In other words, the purified protein is immunologically
It was revealed that it has a structure equivalent to that of ANG2. In order to clarify the amino acid sequence of the carboxy-terminal side of the purified protein, carboxypeptidase Y was applied to the purified protein at varying times, and the released amino acids were quantified (using the carboxypeptidase method). Method for determining the carboxy-terminal amino acid sequence). the result,
-Tyr-Ile-His-Pro-Phe (carboxy terminal)
It was expected that Furthermore, when the purified protein was subjected to acid hydrolysis and then subjected to amino acid analysis, results were obtained that matched the amino acid composition expected from the base sequence. Example 3 Separation of ANG2 from purified and separated DHFR-ANG2 The purified protein obtained in Example 2 (approximately 10 mg,
Approximately 508 nmole of DHFR-ANG2) was freeze-dried,
Dissolve this in 2ml of 70% formic acid and add about 200mg
Bromsyanide was added and dissolved, the mixture was sealed tightly under a nitrogen atmosphere, and the mixture was allowed to react at room temperature with stirring for 24 hours. After the reaction, 20 ml of water was added and then freeze-dried.
The specimen obtained by freeze-drying was dissolved in 10 ml of 30% acetic acid. 0.5ml of it (approximately 25nmole)
(which should contain DHFR-ANG2) was separated using an HPLC device (Shimadzu LC-4A) and an Inertsil-0DS 5 μm column. Elution is a concentration gradient of acetonitrile (15% to 50%) in 0.1% trifluoroacetic acid.
This was done by multiplying. From 0 to 2 minutes,
A linear concentration gradient of 15% to 50% acetonitrile was applied from 2 minutes to 32 minutes using 15% acetonitrile. As a result, an elution curve as shown in FIG. 3 was obtained. The peak approximately 17 minutes after sample injection was collected, and the separated eluate was dried in an evaporator, and a small amount of water was added and lyophilized to remove the solvent to obtain the peptide. The obtained peptide was acid-hydrolyzed,
One-fifth of the volume was used for amino acid analysis. As a result, aspartic acid (Asp), valine (Val), isoleucine (Ile), tyrosine (Tyr), phenylalanine (Phe), histidine (His), arginine (Arg), and proline (Pro) are respectively 2.5, 2.5, 2.3, 2.6, 2.8, 2.5, 2.6, and
2.7 nmole each was detected. The amino acid composition is
The value was consistent with that of ANG2, and the sample used for amino acid analysis contained approximately 2.6 nmole (approximately 2.8 μg).
It was revealed that it contained ANG2. Using this result, by separating 0.5 ml of the standard sample obtained by bromcyan treatment using HPLC, the yield is approximately 52% (2.6x5 nmole/25 nmole).
It is shown that ANG2 can be recovered and that by repeating this operation 20 times, about 280 μg of ANG2 can be obtained from 10 mg of DHFR-ANG2. In addition, the yield of purification of DHFR-ANG2 was approximately 48%.
Since the yield of separation of ANG2 from DHFR-ANG2 is approximately 52%, the isolation yield of the ANG2 peptide portion produced by E. coli is calculated to be approximately 25%. Effects of the invention As mentioned above, the novel recombinant plasmid pANG2
-11 encodes DHFR-ANG2, and E. coli containing pANG2-11 is DHFR-ANG2.
Accumulates and produces large amounts of ANG2 in a soluble state. Furthermore, the produced DHFR-ANG2 retains DHFR enzyme activity and can be easily purified. Furthermore, ANG2 can be easily isolated by treating DHFR-ANG2 with bromide cyanide and then separating it by HPLC. Because of these properties, the present invention is useful for DHFR-ANG2 and the production of ANG2 using the same. Also,
A Kpn site has been newly added to pANG2-11, and it is considered to be useful as an expression vector for a heterologous gene.

[Brief explanation of drawings]

FIG. 1 shows the entire base sequence of pANG2-11, in which only one DNA strand sequence of the double-stranded DNA is written in the direction from the 5' end to the 3' end. The symbols in the figure represent nucleic acid bases; A represents adenine, C represents cytosine, G represents guanine, and T represents thymine. The numbers in the figure are 2 for pANG2-11.
Among the restriction enzyme Clas present in the Cla, the Cla cleavage recognition site near the Hind site, 5′-ATCGAT
-3', the first "A" is counted as number 1. FIG. 2 is a diagram showing the base sequence of the portion encoding DHFR-ANG2 present in pANG2-11 and the amino acid sequence of the protein. The symbols in the figure represent nucleobases and amino acids, A is adenine, C is cytosine, G is guanine, T is thymine, Ala is alanine, Arg
represents arginine, Asn represents asparagine, Asp represents aspartic acid, Cys represents cysteine, Gln represents glutamine, Glu represents glutamic acid, Gly represents glycine, His represents histidine, Ile represents isoleucine, Leu represents leucine, Lys Lysine, Met
represents methionine, Phe represents phenylalanine,
Pro stands for proline, Ser stands for serine, Thr stands for threonine, Trp stands for tryptophan, Tyr stands for tyrosine, and Val stands for valine. The numbers in the figure indicate the numbers counted starting from "A" of the ATG codon that encodes the first amino acid, methionine. Figure 3 shows a high performance liquid chromatogram of a DHFR-ANG2 sample treated with bromocyanide. The horizontal axis represents the time after sample injection in minutes, and the vertical axis represents the absorbance at 220 nm in arbitrary units. The peak indicated by the arrow is the elution peak of ANG2.

Claims (1)

[Claims] 1. A novel recombinant product that is stably replicated in E. coli, can confer trimethoprim resistance and ampicillin resistance to the host E. coli, has a size of 4651 base pairs, and has the DNA sequence shown below. Plasmid pANG2−11. [Table] [Table] [Table] [Table] [Table] 2 E. coli containing pANG2-11. 3. A dihydrofolate reductase-angiotensin fusion protein produced by E. coli containing pANG2-11 and having the amino acid sequence shown below. [Table] 4. E. coli containing pANG2-11 was cultured, and dihydrofolate reductase-angiotensin fusion protein was extracted from the cell-free extract of the cultured cells using dihydrofolate reductase activity as a guideline. 1. A method for separating and purifying a dihydrofolate reductase-angiotensin fusion protein, the method comprising purifying it using bonded affinity column chromatography and anion exchange column chromatography. 5. A method for producing angiotensin, which comprises decomposing a dihydrofolate reductase-angiotensin fusion protein produced by Escherichia coli containing pANG2-11 by a bromcyan degradation method, and then separating and purifying angiotensin.