JPS62130697A - Production of peptide - Google Patents

Abstract

PURPOSE:To obtain a polypeptide having hANP (human atrial sodium diuretic peptide) activity, by cultivating an animal cell transformed with a vector derived from BpV (bovine papilloma virus) into which hANP genome. CONSTITUTION:Plasmid pBRhANP-1 integrated with hANP chromosome gene is isolated from a human gene bank by the use of hANP as a probe. The plasmid is cleaved by BamHI to give ahANP genome fragment. Both the ends of the fragment are changed into HindIII adherent ends and plasmid pBPVhANP-1 inserted into HindIII cite of plasmid pdBPV-1, wherein the DNA fragment is bonded to pML-2 of a derived substance of bovine papilloma virus DNA(BPV-1) and pBR322, is formed. The plasmid is transduced into mouse 127 cell by calcium phosphate method, a cell to form a focus is separated and the cell is cultivated.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing peptides. For more details,
Human atrial natriuretic peptide (abbreviated as hANP)
It can be obtained by culturing animal cells transformed with a plasmid vector derived from bovine biroma virus (abbreviated as BPV) into which the genome has been inserted! Zotide, or α-h, which has relaxing activity on chick rectal smooth muscle causing Vj symptoms
This invention relates to a method for producing a peptide that immunologically cross-reacts with an ANP antibody.

(Problems to be Solved by the Present Invention) Recently, it has been discovered that rat atrial extract has a natriuretic effect (De Bold, A, J, et al.

Life 5clence 28+ 89 + 198
It has been shown that there are peptide-like substances with molecular weights of 20,000 to 30,000 and less than 10,000 that have IJ diuretic effects (Curris, M, G,
et al., 5science 221 +71.1983).

In addition, Kanzuki et al. recently reported that human atrium has a diuretic effect28.
We isolated a peptide consisting of several amino acids, determined the amino acid sequence, and determined the amino acid sequence.
trial natriuretic polypep
(Kangawa, ni, ra r)
Biochem.

Biophys, Res, Commun, , 1
18, 131, 1984). They also revealed that this peptide has a significant diuretic effect (particularly natriuretic effect) as well as a blood pressure lowering effect. Furthermore, they found that β-hANP (α-hAN consisting of 56 amino acids), which has similar natriuretic activity, was extracted from human atrium.
antiparallel γ dimer of P) and some-bANP (α-hAN at the C-terminus)
We isolated and identified the katana e-lipeptide, which consists of 126 amino acids with a P structure, and determined their amino acid sequences (
Kangawa, Ni+ et al., Nature 313.3
97.1985).

On the other hand, the present inventors' group attempted to clone a cDNA fragment encoding this polypeptide with natriuretic effect.
e309s724.1984), and also succeeded in expressing the polypeptide in Escherichia coli (Japanese Patent Application No.
9-116605).

In recent years, growth hormone has been developed using genetic engineering technology.

Many physiologically lubricating substances, such as insulin and interferon, can now be produced by E. coli, but on the other hand, there are some substances that are difficult to produce in E. coli, and some that do not have biological activity even if they are produced. In order to solve this problem, the production technology of useful substances using animal cells as hosts has been attracting attention in recent years. This is because modification of proteins is expected.

In general, many of the useful physiologically active substances produced in the living body of animals become biologically active only after proteins undergo modification, such as post-translational addition of sugar chains, acylation, amidation, and processing. Even if these physiologically active substances are produced in E. coli, they cannot be expected to have the same activity as the natural type.

This is because E. coli does not have the above-mentioned modification function. In addition, animal cells have the characteristic that they can easily secrete proteins or peptides to the outside of the cell, which is difficult for E. coli.

It is extremely important industrially to efficiently produce and supply useful peptides with physiological activities equivalent to those of the natural type. Small molecular weight! In the case of zotide, it is possible to chemically synthesize and supply it, but this is generally difficult when the molecular weight is large.

In view of the above problems and facts, the present inventors have conducted intensive research and have discovered a polypeptide having hANP activity (
A polypeptide that has a relaxing effect on chick rectal smooth muscle or a polypeptide that immunologically cross-reacts with α-hANP antibody) is cultured in animal cells transformed with a BPV-derived vector into which the hANP genome has been inserted. As a result, they discovered scales that can be manufactured and completed the present invention.

(Specific means for solving the problem) First, hANP cDNA, which has already been prepared by the group of the present inventors,
(Olukawa, S. et al. Nature 309
#724.1984) as Zorobe, American Man
iatis human gene puncture (Lawn, R, M, et al.

Ce1l15.1157.1978), hANP chromosomal gene (hANP f: BamHI 3.4
grasmid pB, which incorporates a DNA fragment of kb
Isolate RhANP-1. Next, as shown in Figure 1, pBRhANP-1 was cut with BamHI, and both ends of the hANP danome genome were changed to sticky ends with HlndTI.
Plasmid pdBPV-1 (
5arver+N, et al* Pro, Natl, Aca
d, cilasmid pBPVh inserted into the H1nd11 site of Sei, USA+79.7147.1982).
Create ANP-1. This plasmid contains the entire r genome of bovine habilomavirus (BPV).

In addition, the aforementioned hybrid cilasmid pdBPV-1 can be used to transform mouse cells into cancer (focus formation) by transfection (transformation) using the standard calcium phosphate method.
It is known that (Lowy, D. R., et al.
Nature 287 e 72 +1980).

Next, pBPVhANP-1 is introduced into mouse C127 cells by the calcium phosphate method, and focus-forming (transformed) cells are separated. By culturing these transformed cells, it is possible to obtain a peptide that has relaxing activity on chick rectal smooth muscle or that immunologically reacts with the α-hANP antibody. Both components with diuretic activity obtained from atrial extracts simultaneously have chick smooth muscle relaxing activity (Currie, M.G., et al. +5
science 221 + 1983), and α-
Because it cross-reacts with hANP antibodies, the R peptide produced here has diuretic activity, including γ-hANP and α-hANP! There is no doubt that it is zotide (hANP). In addition, peptides having the above-mentioned activities are
It can be obtained both from the culture solution of the mouse transformant and from within the cells.

Hereinafter, the present invention will be explained in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

〔Example〕

Already cloned cDNA (Oikawa+8.
and others.

Nature 309, 724, 1984) as a glove, human gene punk (Law+R, M,
pBRhANP-1 (a plasmid in which a 34 Kb BamHI DNA fragment containing the hANP genome gene is inserted into the BamHI site of pBR322) screened from pBRhANP-1 (Ce 1115 + 1157.1978) was mixed with 60 units of BamHI at 37°C.
, and was cut by heating for 1 hour. Next, 65℃1
The enzymatic reaction was stopped by heating for 0 minutes, and the pDNA fragment was precipitated by adding 2.5 times the strength of ethanol and leaving it at -20°C overnight. Subsequently, the DNA fragment was mixed with 50mM Tris-HCl (pH 7,2), 10mM
MgSO4,0,1mM dithiothreito
Kle in a solution of 50 ttll (DTT)
By reacting 5 units of nowenzyme (Pehringer Mannheim) at 26°C for 20 minutes, the sticky end of BamI (I break point was made blunt), and after extraction with phenol-chloroform, D was extracted with ethanol as usual.
NA fragments were precipitated. Next, the above DNA fragment was
M Tris-HCl (Pl(7,6), 10mM MgC
A2. T4 DNA + 7 gauze 2.8 in 20 μl of a solution consisting of 0.5 mM ATP, 10 mM DTT.
By reacting for 5 hours at 22°C and overnight at 14°C using a unit, the above DNA fragment was injected with Hl ndII[
A linker (C-A-A-G-C-T-T-G: Takara Shuzo m) was added. The reaction was stopped by heating at 65°C for 10 minutes, and the DNA fragments were prepared in the usual manner. Obtained by precipitation with ethanol.Subsequently, 50 m?if NaC6,1
10mM with 0mM MgCA2.1mM DTT
Hl in 50μ of Tris-HCl buffer solution (PH7,5)
By heating at 37°C for 2 hours using ndlI [(120 units), both ends of the DNA fragment were converted to H1ndlI.
After forming sticky ends, the DNA fragment was extracted and purified by electrophoresis.

Next, 0.4 μg of the above DNA fragment and pdBPV-10,1414 digested with HlndII were mixed with 20 μg of Rider John solution (10 mM MgCA, 2.1 mM A
TP, 2.8 units of T4DN in 20mM) lithium-hydrochloric acid solution containing 5mM DTT, p) 17.6)
It was reacted with A ligase (manufactured by Takara Shuzo Co., Ltd.) at 14°C for 16 hours. 20 μl of this reaction solution was used to transform Escherichia coli HBIOI strain treated with Ca CL2 using a conventional method. Transformants were screened using ampicillin resistance (Apr) as an indicator. Plasmid DNA was prepared from eight transformants using a conventional method, and one of the plasmid DNAs, pBP
VhANP-1 was selected.

2. pBPVhANP-1)? Mouse C127 cells were transfected with 10% beef tallow serum (KA, Bloproduc).
The day before transfection, mouse C127 cells cultured in Gibco's DME (Dulbetzko's modified Eagle) medium containing 5x
Seed at 10 cells and incubate at 37℃, 5% CO2.
Culture was started in an incubator. DNA was introduced into C127 cells as follows.

10 pg of pBPVhANP-1 cilasmid was precipitated with ethanol, 2 XT (BS (pH 7,05)
That is, 10 g of HEPES, NaC41 in 1 bottle.
6g, KCL 0.74ji *NIL2T(PO
Dissolve 40.4g + dextrose 2g,
Adjust the pH to 7.05 with NaOH and sterilize the solution 250μ! 250 μl of 250 mM CaCtz was added dropwise. This solution was heated to 455 ml at room temperature.
After standing still for a minute, it was added to a 6 cm petri dish in which C127 cells were growing. 4.5m after standing at room temperature for 200 minutes
1 of DME medium was added and cultured at 37° C. in a 51 Co2 incubator. After 4 hours, remove the medium and add 4 m
After washing with 1 ml of medium, 1 ml of DME medium containing 25% glycerol was added and left to stand for 1 minute. Except for this liquid,
After washing twice with 5 ml of PBS(-), 5 ml of DME
A medium was added and cultured at 37°C in a 5% CO2 incubator. The next day, remove the medium and use 5ml! P of
After washing twice with BS(-), 0.5ml! Trizocine-EDTA solution (trypsin 0.125%).

EDTA O, 01%) was added, and the mixture was allowed to stand at room temperature for 3 to 4 minutes.

After adding 5 TLl of culture medium and disintegrating the cells, the cells were centrifuged at 1000 rpm for 5 minutes to pellet the cells. Dispense the sediment into 3 ml of medium.
Each sample was added to 6 crn bottles with 4 ml of medium added. Cells were cultured at 37°C in a 5% CO2 incubator, and the medium was replaced every 3 days. The foci that appeared after approximately 3 weeks were isolated and placed in a 24-well plate at 0.000.
Culture was performed in 5 ml of DME medium.

3. Biological activity of the product of the transformed cell The biological activity of the product of the transformed strain (the clone that formed the focus in Example 2) was determined using the method of Currie et al.
rrie+MG, et al., 5science 221 +
71 + 1983), the relaxing activity of chick rectal smooth muscle was used as an index. In addition, α-hANP has been isolated so far.

γ-hANP, a peptide with a similar primary structure isolated from rat atrium, is already known to have diuretic activity as well as relaxant activity in chick rectal smooth muscle (
Kangawa, Niira Blochem, Blop
Hys, Res.

Commun, 118, 131.1984 and Cu
rrie, M.G.

et al., 5science 221, 71.1983). Hereinafter, the product obtained in this example that has relaxing activity on chick rectal smooth muscle will be abbreviated as ANP.

Chick rectal smooth muscle relaxing activity (hereinafter simply referred to as relaxing activity) was examined using the method described above for 150 μl of the culture solution of the 9 focus-forming clones obtained in Example 2 (transformation 1). As a result, as shown in Table 1, although there were differences among the clones, relaxation activity was observed in the culture fluids of all clones. No relaxing activity was observed in cells transformed with DNA (pdBV-1) that did not contain the hANP chromosomal gene, which was used as a control. still,
The strength of the relaxing activity shown in Table 1 is α-hANP 10
pmol, 20 pmol activity is used as standard.

Next, regarding clone A9, which is one of the clones with strong relaxing activity in Table 1, Macphersonel,
The method of A. et al. (Vlrology 23, 291,
(1964), subcloning was carried out in 0.3-t soft agar, and the following experiments were further conducted on the obtained clone A9-14.

4. Analysis of product (ANP) First, the diurnal changes in ANP production of clone 9-14 were analyzed by radioimmunoassay (R) using α-hANP antibody.
IA) (Mlyata et al., Bloche
rn.

Biophys, Res, Coynmun, , 12
9, 248, 1985).

Clone A9-14 was added to 6α Sheare 5.5 x 1.
Inoculate at a concentration of 0.05 cells/5 ml and add medium (
DME medium) was replaced, and ANPR secreted into the medium was measured by the RIA described above. As a result, as shown in Figure 2, this clone constantly maintained A over 25 days.
NP (in this case a substance that cross-reacts with the α-hANP antibody) continued to be secreted into the medium. On the other hand, hANP as a control
No ability to produce ANP was observed in the clone (clone 68) transformed with pdBV-1 that does not contain the gene.

Next, clone A 9-14 was cultured in large quantities (approximately 4 × 10 calls) while changing the medium every 3 days.
L. Each culture supernatant was collected and the products were analyzed as follows.

Add acetic acid to the culture supernatant of 1.71 so that it becomes IN,
After processing at 00°C for 5 minutes and returning to room temperature, the supernatant was subjected to LC-
Buffer A (water niacetonitrile: 10% trifluoroacetic acid = 90:10:1) was adsorbed onto an 8orb ODS column (manufactured by Chemco) using a patch method. Elution was performed using 200 d of acetic acid (40:60:1). After drying 1 ml of it under reduced pressure and dissolving it in IN acetic acid, it was subjected to reverse phase HPLC (TSKODS120A: manufactured by Toyo Kodatsu Co., Ltd., column size: 4.6 x 250 mm).
After equilibration with buffer, it was eluted with a linear concentration gradient solvent from buffer A to buffer B (flow rate:
1.5m/! /min.

Fraction size: 1.5+++l). For each elution fraction,
p ANP i was measured by RIA as described above. The elution pattern and RIA measurement results are shown in FIG. As shown in Figure 3, the main RIA active fractions are divided into three fractions: γ fraction (retention time around 35 minutes), β fraction (retention time around 29 minutes), and α fraction (retention time around 22 minutes). Admitted.

On the other hand, AN present in cultured cells of clone A9-14
P was also analyzed as follows. Approximately 1.6×10
32 ml of IN acetic acid was added to the cultured cells, homogenized, and then 64 d of chilled acetone was added.
After centrifugation at Xg for 30 minutes, the supernatant was dried under reduced pressure. Approximately 1/10i1 of it was dissolved in IN acetic acid, fractionated by reverse phase HPLC under the same conditions as above, and each fraction was analyzed by RIA.
I put it on. As a result, as shown in Fig. 4, the peak of RIA activity was α, similar to that of the culture supernatant.

It was observed in three locations, β and γ. Figure 5 shows the culture supernatant (4
) and the elution/lean of the intracellular (B) RLA active fraction.

Vector containing no hANP chromosomal gene (pdBV-
No product exhibiting RIA activity was observed in the clone transformed by 1), indicating that the RIA activity fraction described above is a product from the hANP chromosomal gene. In addition, diuretic activity in rats for both α, β, and γ was determined by Kangava et al.
ophys, Res, Common, + 118 +
131 +1984), all of them were found to have biological activity.

5. Product identification Among the elution fractions of reverse phase HPLC shown in Figure 3, the standard α
-Products were identified for both periods corresponding to the retention time of hANP (22 minutes). That is, the two fractions were further subjected to reverse phase HPLC under the same conditions as described above except that the concentration of acetonitrile in buffer B was changed to A, and the elution fraction corresponding to a retention time of 22 minutes was collected. Using an analyzer (Hitachi Model 835-50), the amino acid sequence of the amino terminal part was determined using a gas phase protein sequencer Mod.
el 470 A (Applied Biosystem
(manufactured by m company) according to a conventional method.

As a result, the amino acid composition values were in good agreement with the α-hANP peptide consisting of 28 amino acids, and the amino terminal was S
er -Leu -Arg -Arg -Ser -S
er -(Cys) -Phe -Gly -Gly
It was recognized that --=. This result shows that α-hA
Np-e peptide is further synthesized from a precursor protein produced through processing of hANP chromosomal gene mRNA (it is known that the hANP chromosomal gene has an intron) in animal cells (in this case mouse cells). This indicates that it was generated through processing.

Although there are no amino acid analysis results, in Figure 3, there is a high RIA at the position corresponding to the retention time of standard γ-hANP.
Since there are fractions showing activity, it is predicted that these peptides are also produced in the above-mentioned clones.

[Brief explanation of drawings]

Figure 1 shows the expression vector p containing the hANP chromosome.
An outline of the method for producing BPVhANP-1 is shown. Figure 2 shows
14) and the clone (clone 68) into which pdBV-1 was introduced. Figure 3 shows
Figure 4 shows the elution of the culture supernatant of clone A9-14 using reverse phase T (PLC) and the elution of immunoreactivity.
and turns respectively. Figure 5 shows clone A 9-14.
Figure 3 shows the immunoreactivity pattern corresponding to the reverse phase HPLC elution of the culture supernatant and intracellular extract (time indicates the retention time in reverse phase HPLC).

Claims (1)

[Claims] 1. Obtained by culturing animal cells transformed with a plasmid vector derived from bovine papillomavirus (BPV) into which the human atrial natriuretic peptide (hANP) genome has been inserted; A method for producing a peptide having relaxing activity on chick rectal smooth muscle. 2. The production method according to claim 1, wherein the peptide having relaxing activity immunologically cross-reacts with the α-hANP antibody. 3. The above vector is bovine papillomavirus (BPV)
2. The production method according to claim 1, characterized in that it has the entire genome of. 4. The production according to claim 1, wherein the human atrial natriuretic peptide (hANP) genome is a genome derived from a DNA fragment obtained by cleaving human chromosomal DNA with the restriction enzyme BamHI. Law. 5. The manufacturing method according to claim 1, wherein the animal cells are mouse cells C127. 6. Claims 1 to 6, wherein the amino terminal amino acid sequence of a peptide having relaxing activity on chick rectal smooth muscle or a peptide that immunologically cross-reacts with an α-hANP antibody is [there is an amino acid sequence]. The manufacturing method described in Section 2. 7. Claims 1 and 2, characterized in that the peptide having relaxing activity on chick rectal smooth muscle or the peptide that immunologically cross-reacts with the α-hANP antibody is α-hANP. manufacturing method.