JPH0691833B2 - Method for producing polypeptide having diuretic effect - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing the above-mentioned polypeptide, which utilizes a novel gene (DNA) encoding a polypeptide having a diuretic action newly found in human atrium.

[0002]

2. Description of the Related Art It is known that human atria contain granules morphologically similar to those found in peptide hormone-producing cells (JD Jamieson & GEPalade, J.
Cell Biol., 23 , 151, 1964). Recently, rat atrial homogenate and its granules have been known to cause natriuresis in rats (AJ DeBold et al., Life S.
ci., 28 , 89, 1981, R. Keeler, Can.J.Physiol.Pharmaco
l., 60 , 1078, 1982, etc.), and the presence of peptide-like substances having a natriuretic effect of 20,000 to 30,000 and 10,000 or less in the atrium of human, rabbit, pig and rat. (G. Mark, S. Currie et al., Science,
221 , 71-73, 1983).

More recently, Flynn, TG et al. Isolated a peptide of 28 amino acids having a diuretic effect from rat atrium (Biochem. Biophys. Res. Commun.,
118 , 131-139, 1984). Also, Kangawa, K. and Matsu
o, H. found a peptide consisting of 28 amino acids having similar activity in human atrium and named it α-hANP. And α-hANP is H-Ser-Leu-Arg-Ar
g-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-
Ala-Glu-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH
(Cys at position 7 and Cys at position 23 are disulfide-bonded), and it was clarified that they have a remarkable diuretic action as a pharmacological activity, especially a natriuretic action and a hypotensive action (Biochem. Biophys. .Commun., 1
18 , 131-139, 1984, and JP-A-60-13659.
6).

On the other hand, it is known that various physiologically active peptides are not directly produced in vivo but their precursors are produced by the action of endopeptidase or exopeptidase. For example, β
-Endorphin and γ-LPH have been reported to be produced with β-LPH as a precursor and β-MSH with γ-LPH as a precursor (Takahashi, H. et al., FEBS Lett. 1) .
35 , 97-102, 1981). We note this,
Assuming that α-hANP is also produced from a certain precursor, the gene encoding α-hANP and the mechanism of its production were studied intensively.

As a result, we succeeded in isolating and identifying a gene (DNA) encoding a peptide (hereinafter abbreviated as γ-hANP) which is a precursor of α-hANP.
The present invention has been completed by creating a recombinant DNA having NA and finding that a peptide having a diuretic action, particularly a natriuretic action and a hypotensive action can be produced by using the recombinant DNA.

[0006]

That is, the present invention is based on α-h
Α-hAN by culturing a transformant having a DNA containing a nucleotide sequence encoding ANP and its recombinant DNA
P and its precursor (eg, γ-hANP; this amino acid sequence is shown in FIG. 1) and a method for mass-producing various polypeptides having the same biological activity as those of P and its precursor.

Α-hA isolated from human atrial granules
The structure and pharmacological activity of NP precursor γ-hANP are described in detail in JP-A-60-260596.

[0008]

The present invention will be described in detail below. (1) Identification of γ-hANP gene that is a precursor of α-hANP (see SEQ ID NO: 1) A gene having a nucleotide sequence encoding α-hANP was prepared as follows. First, poly ARNA obtained from human atrial granules using oligo dT column
(MRNA) to Okayama-Berg method (Mol. Cel
l.Biol., 2, 161-170, 1982) to prepare a cDNA library, and then the 12th (Met) to 16th (Gly) of the amino acid sequence of α-hANP (described above).
A chemically synthesized 14-mer oligonucleotide corresponding to is used as a mixed probe,
A clone that hybridizes with the above probe labeled with ³² P was selected.

As a result, 23 target clones were obtained from about 40,000 transformants (the above-mentioned cDNA library). For the plasmids from 8 of these clones, the 14-mer mixed probe described above was used.
When the nucleotide sequence by the dideoxy method was examined using e as a primer, all plasmids had a nucleotide sequence encoding α-hANP. Of these, two plasmids with long insert DNA fragments, phANP
1 and phANP82 (about 950 bp and about 850 bp, respectively)
(Comprising the following) was determined. The nucleotide sequence is Maxam-Gilbert (Meth. Enzym., 65, 499 to 560, 1
980) and Sanger et al. (Proc. Nath. Aced. Sci. USA, 74 ,
5463-5467, 1977).

Both DNAs have a long open reading frame (translatable region) starting from the translation initiation codon ATG and ending at the translation termination codon TGA, and one is shorter than the other by about 100 bp in the 5'-unsided region at all. Had the same sequence. In this translatable region, α-hANP (Kangawa, K. & Matsuo, H., Bioche, which consists of 28 amino acids whose amino acid sequence has already been clarified,
m.Biophys.Res.Commun., 118 , 131-139, 1984) was present (see SEQ ID NO: 1).

On the other hand, a polypeptide having a diuretic effect (γ-hANP) purified from human atrial granules is
The end is H-Asn-Pro-Met-Tyr-Asn -... and the C-end is -Asn-Ser.
-Phe-Arg-Tyr-OH was revealed to consist of 126 amino acids (Fig. 1 and JP-A-60-2605).
96). This result and the above-mentioned phANP1
And from the base sequence on phANP82, γ-h
It is concluded that the gene and amino acid sequence encoding ANP (FIG. 1) is as shown in SEQ ID NO: 1 (showing the sequence cloned in phANP82). In the figure, 1 to 25 of the amino acid sequence are considered to be signal peptides involved in protein secretion, and a polypeptide consisting of 126 amino acids starting from Asn at the 26th position and ending at Tyr at the 151st position is γ-hANP ( The amino acid sequence consisting of the signal peptide and γ-hANP is shown in FIG. 2).

Interestingly, the amino acid sequence of α-hANP (from the 124th position to the 151st position in the figure) is γ-hA.
It exists on the C-terminal side of the amino acid sequence of NP. This explains the results disclosed in JP-A-60-260596 without contradiction. Thus, as initially expected, the precursor of α-hANP, namely γ-h
It was possible to confirm the presence of ANP and to isolate its gene.

The DNA shown in SEQ ID NO: 1 and its DNA fragments shown in FIGS. 3 and 4 are α-hANP, γ-hANP, and biologically combined with them, as described below. It is obvious that it is industrially useful as a material for mass-producing various polypeptides having the same activity (particularly diuretic activity and hypotensive activity).
Also, a peptide having a diuretic effect (β-hANP), which was also found in human atrium, has been suggested to be a dimer of α-hANP (JP-A-60-184098).
Also, the usefulness of the precursor and its gene can be seen.

In order for α-hANP to be produced using γ-hANP as a precursor, it is necessary to cleave the C-terminal of pro-arg (amino acid sequence of 123-124 in SEQ ID NO: 1). Is produced by cleavage of gastrin-releasing hormone (GRP) at the C-terminus of the pro-arg sequence present in its precursor in the mammalian intestine and spinal cord (Reave, JR et al., J. Biol. Chem. ., 2
58 , 5582-5588, 1983 and Minamino, N. et al., Biochem.
Biophys.Res.Commun., 119 , 14-20, 1984), α-hA
NP also has similar processing in vivo (probably enzymatic action)
Therefore, it is considered to be generated from γ-hANP. (2) Preparation of recombinant DNA γ-hANP containing a nucleotide sequence encoding α-hANP
As described in detail below, the recombinant DNA of the gene was prepared by cloning the DNA fragment containing the γ-hANP gene obtained as described above into M13 DNA and
Vitromutation method (Zoller, MJ & Smit
h, M., Nucl.Acid.Res. 10 , 6487, 1982)
-A restriction enzyme EcoRI cleavage site and translational codon (ATG) are added immediately upstream of the hANP gene, and an appropriate expression control nucleotide sequence region (including promoter and SD sequence) is inserted upstream of the cleavage site. Made by.

First, at the PstI cleavage point of M13mp8RFDNA (manufactured by PL), phAN obtained in (1) above.
A DNA fragment (about 700 bp) containing the γ-hANP gene obtained by cleaving P1 with PstI was inserted into E. coli (J
M103) was transformed. Next, the phage DNA (single-stranded DNA) in which the desired DNA fragment (about 700 bp) was inserted was separated from the culture solution of the above-mentioned transformant, and in vitro mutation (in vitro mutat) was performed.
ion) (site directed mutati)
(also called on) method was used to insert an EcoRI cleavage recognition site (GAATTC) and ATG (triplet codon encoding Met) immediately upstream of the γ-hANP gene.

The in vitro mutation is Eco
After forming a hetero duplex of chemically synthesized single-stranded DNA having a sequence of ATG immediately 3'to the RI cleavage recognition site and the above-mentioned single-stranded phage DNA, the conventional method (Zoller, JM & Smith, M., Nucl.Aci
d.Res. 10 , 6487, 1982) DNA polymerase IK
E. coli (JM1
03) was transformed.

The target nucleotide sequence (SEQ ID NO: 1, 6
7th to 75th nucleotide sequence ACC AGA G
(Base sequence in which CT is converted to GAA TTC ATG)
The plasmid having D is obtained from the above transformant strain.
NA (RFDNA) was selected by analysis by digestion with a restriction enzyme (EcoRI), and the region around the base sequence was further subjected to dideoxy chain termination.
Ion method (Sanger, F. et al., Proc. Natl. Acad. Sci. USA, 74 ,
5463-5467, 1977). This plasmid is M13mp8-hANP52
It was named 5 and was used in the next experiment.

The cloning of the γ-hANP gene into an expression vector is carried out by the above plasmid M13mp8-hANP.
525 to EcoRI and SalI (SalI cleavage site is M
Approximately 510 bp obtained by digestion with 13mp8 DNA)
Of the plasmid pS20 (see FIG. 5) which had been constructed by the present inventors.
It was done by substituting the 11 fragment (about 450 bp).

The plasmid pS20 contains the λPL promoter and the SD sequence of the λphage CII protein gene (Shine-
Dalgano sequence) as a 5'untranslated region, and the foreign gene inserted downstream (EcoRI breakpoint) of the region is λP
The E. coli transformant strain N4830 / pS20, which is a plasmid constructed so that it can be expressed under the control of the L promoter, is named SBM271 and has been deposited with the Microindustry Research Institute under the Budapest Treaty (FER
MBP-535). The plasmid p constructed in the present invention
S220 (see FIG. 5) is E. coli N4830 according to a conventional method.
Strain, and the transformed strain (N4830 / pS2
20) was investigated for the production of γ-hANP.

The production of γ-hANP is carried out by shifting the culturing temperature of the transformant strain from 32 ° C. to 42 ° C. during the culturing, and then adding ³⁵ S-methionine to pulse-label the produced protein (2 minutes). Then, the labeled produced protein is SD
It was checked by detection by S-PAGE (SDS polyacrylamide gel electrophoresis). as a result,
A clear protein band not seen in the pS220-free strain (N4830) was observed only in the culture of the transformed strain (see FIG. 6).

Furthermore, from the λPL promoter region of the plasmid pS220, the translation initiation codon (A of γ-hANP) (A
The region between TG) is the S of the Escherichia coli outer membrane protein (lpp) gene
Similar protein production was also observed in the plasmids (pS223-3 and pS224-3: see FIG. 7, respectively) converted into a region containing the D sequence and the SD sequence of the MS2 phage A protein gene (see FIG. 6).

The protein band was found at a position larger than the theoretical molecular weight of γ-hANP of 13,679 (about 20 Kd), but γ-hANP purified from human atrium was also SDS-.
Since it migrates to the same position by PAGE, the band of the protein is γ produced by the transformant N4830 / pS220.
-Determined to be hANP. In SDS-PAGE, it is a common phenomenon that proteins migrate to positions larger than the theoretical molecular weight.

Further, this means that a chemically synthesized linker having a translation stop codon designed in three different reading patterns at the end of the γ-hANP structural gene:

[0024]

[Chemical 1] In the same plasmid with the addition of
This was also supported by the fact that a protein band not found in the control was seen at the position. This result denies the possibility that the translation termination codon TGA (codon immediately downstream of the 151st amino acid Tyr in SEQ ID NO: 1) was incorrectly translated in vivo and a large whitening could occur. Is.

Thus, it was confirmed that γ-hANP which is a precursor of α-hANP can be produced in a microorganism by using the gene obtained in the present invention. Here, γ-
hANP signal peptide portion (SEQ ID NO: 1 to 1
25 amino acid sequence) except the amino acid sequence (26-1
51) was described as a recombinant DNA having a nucleotide sequence that encodes a polypeptide containing a signal peptide (amino acids 1 to 151 in FIG. 2 and SEQ ID NO: 1) (FIG. 4), or α-hANP. (SEQ ID NO: 1
It is clear that a recombinant DNA can be prepared by the same method for the nucleotide sequence encoding a polypeptide starting from an appropriate position (124 to 151). In addition, here, mR obtained from human atrial granules
Although NA cDNA was used as the gene for γ-hANP, the gene encoding the amino acid sequence may be chemically synthesized.

Furthermore, in the construction of recombinant DNA, γ
An example was shown in which the λPL promoter was used to express the -hANP gene, but the promoter region is not limited to this. For example, when expressed in E. coli, tryptophan (trp), lactose (lac), lipoprotein (lpp), alkaline phosphatase (PHOA).
PHOS and the like) and promoter regions such as outer membrane protein (omp) gene, and a hybrid promoter (tac promoter) of tryptophan and lactose promoter may be used.

In the case of expression in yeast, alcohol dehydrogenase (ACH), glyceraldehyde dehydrogenase (GAP-DH), phosphoglycerokinase (RGK) and inhibitory acid phosphatase (PHO 5 ) Etc., and in the case of animal cells, the early or late promoter region of SV40 can also be used.

Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.

[0029]

[Example] 1. Construction of α-hANP cDNA library
Isolation and Identification of Gene 1-1 Construction of cDNA Library From two human atria isolated from a 82-year-old female and a 61-year-old male, the method of Chirgwin et al. (Chirgwin, JM
Et al., Biochemistry 18 , 5294-5299, 1879) 4M
1 mg of RNA was extracted with guanidinium thiocyanate. Then 0.5 M LiCl ₂ , 10 mM EDTA
And 75 μg of poly (A) ⁺ RNA (mRNA) bound to oligo dT cellulose were separated using 10 mM Tris-HCl buffer (pH 7.2) containing 0.5% SDS as a binding buffer (Nakazato, H. & Edmonds, DS, Meth.Enzym., 29 ,
431-443, 1974).

Subsequently, the method of Okayama-Berg
(Mol.Cell.Biol., 2 , 161-170, 1982), 15 μ
A cDNA library (plasmid) was prepared by using g of poly (A) ⁺ RNA and 4.2 μg of vector primer DNA, and Escherichia coli WA802 strain was transformed. Transformants were screened on LB-agar medium containing 40γ / ml ampicillin to give poly (A)
⁺ About 40,000 transformants showing ampicillin resistance were obtained per ¹ μg of RNA. Separation of 1-2 α-hANP clone About 40,000 colonies obtained in the above were replicated on a nitrocellulose filter, and cultured on an LB agar plate containing 40 γ / ml ampicillin at 37 ° C. for 6 hours and then 180 γ / It was transferred to an LB agar plate containing ml of chloramphenicol and incubated overnight at 37 ° C. Next, after lysing the colonies formed on the filter with 0.5N NaOH
Neutralize to pH 7.0, filter 3 × with 0.5M Tris-HCl buffer (pH 7.0) containing 1.5M NaCl.
SCC (0.15M NaCl, 0.015M Sod
citrate) for 5 minutes each. Finally, after removing the bacterial debris with a paper towel and air-drying, baking was performed at 80 ° C. for 2 hours.

Subsequently, two kinds of chemically synthesized oligonucleotides labeled with ³² P at the 5'end (FIG. 8) were used as mixed probes to carry out hybridization with the DNA molecules immobilized on the above filter (Grunstein). , M. & Hogn
ess, DS, Pyoc.Natl.Acad.Sci.USA, 72 , 3961-3965,
1975). The above-mentioned probe encodes Met-Asp-Arg-Ile-Gly (amino acid sequence from 135th to 139th in SEQ ID NO: 1) in the amino acid sequence of α-hANP.
It is a mixed probe of 14 mer oligonucleotides complementary to RNA, the 5'end of which is [γ. ³² P] AT
Labeled with P and T4 kinase, specific activity 1-2x1
0 ⁶ cpm / pmol.

Hybridization is performed with 1 × Denh.
ardts. [0.2% BSA (Almor Co.),
0.2% Ficol (Sigma) and 0.2% polyvinylpyrrolidone (Wako Pure Chemical Industries)], 0.1%
SDS and 50 μg salmon testis DNA per ml (P
L company) in 3 × SCC at 38 ° C. for 16 hours.
Next, filter the filter with 3 × SCC containing 0.1% SDS.
At 38 ° C., air dried, and exposed to X-ray film. This operation yielded 85 positive clones.

Next, two kinds of probes (probe I and pro
Probe II: Figure 8) separately at 40 ° C and 38 ° C respectively.
Using the same procedure as described above,
Ronnie hybridization was performed. as a result,
It does not hybridize with probe I and does not hybridize with probe II.
Twenty-three clones that hybridize were obtained. continue
Plasmid DN from 8 of 23 clones
A is separated according to a conventional method, and probe II is used as a primer.
The used dyeoxy chain terminator
Ion method (Sanger, F. et al., Pyoc.Natl.Acad.Sci.USA,7
Four, 5463-5467, 1977).

As a result, α-
Since the nucleotide sequence corresponding to the amino acid of hANP was present, the above 8 clones were cDNA of α-hANP.
It was confirmed that the transformant had a plasmid containing Of these plasmids, two plasmids (phANP1 and phANP82) into which the long γ-hANP cDNA had been inserted were selected, and the nucleotide sequence of the inserted cDNA was determined. About 950 bp and 850 bp of cDNA were inserted in phANP1 and phANP82, respectively.

The restriction enzyme cleavage site of the inserted DNA region and the strategy for determining the base sequence are shown in FIG. In the figure, the direction of nucleotide sequence determination is indicated by an arrow, the solid line indicates the upper strand (upper strand) of the double-stranded DNA, and the dotted line indicates the lower strand (lower strand). The nucleotide sequence is based on the Maxam-Gilbert method (Meth. Enzym., 65 , 499 to 560, 198).
0) and Sanger et al. (Proc. Natl. Acad. Sci. USA, 7
4 , 5463-5467, 1977).

In FIG. 9, A is the Maxam-Gilbert method, and B is 5'and 3'labeled with ³² P at 5'and 3 ', respectively.
-GATCGATCTGCCCTC-3 'and 3'-CTAGCTAGACGGGAG-5' using the chemically synthesized oligomers as primers by the method of Sanger et al., And C is the three DNAs obtained by cleaving the plasmids (phANP1 and phANP82) with PstI and PvuII. The fragment was transformed into plasmid pUC8 (Vieir
a, J. & Messing, J. Gene 19 , 259-268, 1982), and each was determined by the method of Sanger et al.

As a result of the above nucleotide sequence determination, both nucleotide sequences of the inserted DNA were in perfect agreement with each other except that the length on the 5'-terminal side was different. Among them, the base sequence of the DNA inserted in phANP1 and the amino acid sequence corresponding to the base sequence are shown in SEQ ID NO: 1. In the sequence of the inserted DNA, there is a long translatable sequence (open reading frame) starting from ATG and ending at the stop codon TGA.
A DNA sequence encoding α-hANP was found on the terminal side (C-terminal side in the amino acid sequence).

Γ-hAN purified and identified from human atrium
Since the N-terminal of P is H-Asn-Pro-Met-Tyr-Asn -... and the C-terminal is -Asn-Ser-Phe-Arg-Tyr-OH (see JP-A-60-260596). 5), the amino acid sequence of γ-hANP is judged to consist of 126 amino acids starting from Asn at position 26 and ending at Tyr at position 151 in FIG. This result is consistent with the amino acid composition and amino acid sequence of γ-hANP purified from human atria.

From the structure, the amino acid sequence of 1-25 starting with Met and ending with Ala in SEQ ID NO: 1 is considered to correspond to a signal peptide involved in secretion. Further, in the base sequence of SEQ ID NO: 1, AATAAA which is generally found upstream of the poly A sequence of eukaryotic mRNA was present near the 3'end. 2. Construction of γ-hANP gene expression vector 2-1 Insertion of hANP gene into M13 DNA M13 mp 8 RF DNA (PL) 0.44 μ
g to 20 μl of Medium-salt Buffer
r (50 mM NaCl, 10 mM MgCl ₂ , 1 mM D
10 mM Tris / HCl buffer containing TT (pH 7.5)
Cleavage was carried out by heating with PstI (16 units) in at 37 ° C. for 1 hr.

Next, the enzymatic reaction was stopped by heating at 65 ° C. for 10 minutes. On the other hand, 20 μg of the plasmid phANP1 DNA obtained from the cDNA library was treated with 100 μl of medium salt buffer in the same manner.
After digestion with PstI (160 units) in r at 37 ° C. for 1 hour, the gel corresponding to a DNA fragment of about 700 bp was cut out by 1% agarose gel electrophoresis and subjected to electrophoresis (electro-elute method). The DNA fragment was extracted and purified by

Next, 20 μl of a ligation solution (10 mM MgCl ₂ , 1 mM ATP, 20 mM Tris · HCl buffer containing 5 mM DTT) of the above two kinds of DNA fragments (66 μg of PstI-digested M13 mp 8 RFDNA and 1 μg of a DNA fragment of about 700 bp) was added. pH 7.6)
5.6 units of T4 DNA ligase (Takara Shuzo) and 14 ℃
And reacted for 16 hours. 20 μl of this reaction solution was used as C
E. coli JM103 50 mM CaCl treated with aCl _2
Two suspensions were added and transformation was performed. Selection of transformed clones was performed as follows.

YT soft agar medium (0.6% containing X-Gal and IPTG kept at 45 ° C.)
100 mM IPT in 3 ml of agar, 0.8% bactotryptone, 0.5% yeast extract, 0.5% NaCl.
G 10 μl, 2% X-gal 50 μl and logarithmic growth phase JM103 suspension 0.2 ml) and appropriately diluted CaCl ₂ suspension 0.3 ml described above and YT.
Agar medium (1.5% agar, 0.8% bactotryptone,
It was spread on 0.5% yeast extract, 0.5% NaCl) and cultured at 37 ° C. for 16 hours. Ten clones were selected from the resulting white plaques, and 2 × YT liquid medium (1.6% bactotryptone, 1% yeast extract, 1.0% NaC) was selected.
After inoculation into 1), the cells were cultured at 37 ° C. for 8 hours. Then 1 ml of the culture
Was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was recovered as a phage solution. The phage DNA [single chain DN
A (ssDNA)] was separated and purified as described below.

2.5 μN was added to 800 μl of the above phage solution.
20% polyethylene glycol containing aCl (PEG6
000) to 200 μl and left at room temperature for 15 minutes 1
Phages were precipitated by centrifugation at 0000 rpm for 5 minutes. Precipitate this phage with TE buffer (1 mM E
DTA, 10 mM Tris HCl buffer pH 8.0) 1
Dissolve in 00 μl, add 50 μl of water-saturated phenol, and add 5
After vigorous mixing for 1 minute, centrifugation was performed at 10,000 rpm for 5 minutes.

80 μl was taken from the aqueous phase and 3M vinegar was added to it.
Sodium acidate (pH 8.0) 3 μl, and ethanol 20
After adding 0 μl and cooling at −70 ° C. for 10 minutes, 1
D by centrifuging at 10,000 rpm for 10 minutes
NA was precipitated. Wash the precipitated DNA once with ethanol
Then, it was dissolved in 50 μl of the above TE buffer. 10 clones
Didyoxy chain among phage DNA
erniation method (Methods in Enzymology6
Five, 560 ~ 580, 1980 Academic Press, New York)
The lower strand (mRN
DNA fragment having a base sequence complementary to A) is inserted.
The two clones that were selected were selected. This phage DNA
The in vitro mutation (in v
used as template DNA for
It was2-2 EcoRI by in vitro mutation
Insertion of cleavage site and translation initiation codon 36mer chemically synthesized DNA fragment with phosphorylated 5'end
Piece (5′-CTCCTAGGTCAGGAATTCATGAATCCCATGTACAAT-3 ′)
Obtained in (2-1) above in a 2 μl solution containing 10 pmol.
5 μl of single-stranded phage DNA solution and 0.1 mM
gCl₂, 0.2M Tri with 0.5M NaCl
Add 1 μl of s · HCl buffer (pH 7.5) and 2 μl of water
Eat at 65 ° C for 5 minutes in a total of 10 μl of solution at room temperature
It was left for 10 minutes.

Next, 0.1M MgCl 2 is added to this mixed solution.
0.2M Tris.HCl buffer containing ₂ (pH 7.
5) 1 μl, 0.1 M DTT 2 μl, 10 mM A
1 μl of TP, 10 mM dATP, dGTP,
2 μl of dCTP and dTTP, 2 μl of water, and DNA polymerase I Klenow-fragment (Klenow f
ragment) (Behring-Mannheim) 5 units and T ₄ DNA ligase (Takara Shuzo) 2.8 units were added and reacted at 15 ° C. for 16 hours. This reaction solution 20μ
E. coli JM103 was transformed with 1 as described above.

Next, as described above, 48 clones were selected from the plaques formed on the YT soft agar medium, inoculated into 2 × YT liquid medium and cultured at 37 ° C. for 8 hours. 1 ml culture
Was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was recovered as a phage solution. On the other hand, from the bacterial cells obtained as a precipitate, an alkaline extraction method (Birnboim, HC & Doly, J. Nucl, Acid, Re
s 7 , 1513-1523, 1979) to extract and separate RF DNA.

Subsequently, the RF DNA was added to EcoRI buffer (50 mM NaCl, 100 mM containing 5 mM MgCl _2).
Tris / HCl buffer pH 7.5) 20μ
3 using EcoRI (Takara Shuzo) 4.2 unit
It decomposed by heating at 7 ° C for 1 hour. The reaction solution was subjected to 2% agarose gel electrophoresis and DN of about 530 bp was applied.
A clone was selected such that an A fragment was generated. (1 clone out of 48 clones) This clone was cloned into M13mp8-hA
It was named NP 525.

Next, 0.4 ml of JM 103 culture solution infected with the phage clone was added to 400 ml of 2 × YT liquid medium.
And 4 ml of non-infected JM 103 culture solution was added and the mixture was kept at 37 ° C for 12 hours
After culturing, the cesium chloride-containing density gradient centrifugation containing ethidium bromide from the infecting bacteria was performed according to a conventional method to obtain RF DNA.
On the other hand, phage DNA was obtained from the supernatant phage solution by the above-mentioned method, and then dideoxy chainer was obtained.
The nucleotide sequence was determined using the mination method (described above). As a result, the expected nucleotide sequence, that is, γ-hA
Immediately before the N-terminal of NP, translation initiation codons ATG and ECO
Sequence GAATT with RI cleavage recognition site GAATTC
It was confirmed that CATG was inserted. Construction of 2-3 γ-hANP gene expression vector (Fig. 5
And FIG. 7) The plasmid pS20, which is a material for constructing the γ-hANP gene expression vector, has already been constructed by the present inventors, and an Escherichia coli transformant strain N4380 having the plasmid has been constructed.
/ PS20 is named SBM 271 and made an international deposit under the Budapest Treaty with the National Institute of Fine Arts and Machines and deposit number FERH BP
I got -535. pS20 is a plasmid capable of expressing a foreign gene under the control of the P _L promoter of λ phage, and is constructed as follows.

First, a 2.4 Kb DNA fragment containing a λP _L promoter obtained by cleaving bacteriophage λCI857 with BamHI and HindIII was inserted between HindIII and BamHI of pBR322 to obtain a plasmid (this plasmid is Nature). 292, 12
8, 1981, which is equivalent to the plasmid pKO30 described in the above). Next, at the HpaI cleavage site of this plasmid, the HaeIII DNA fragment (N of the bacteriophage λcy3048 (obtained from Dr. Hiroyuki Shimatake, Toho University School of Medicine))
A 1.3 Kb fragment containing utR, tR ₁ CII and a part of O) was inserted so that CII was located in the same direction as the transcription direction of λP _L (pS9).

Subsequently, this plasmid (pS9) was added to Bgl
A 0.65 Kb DNA fragment obtained by digestion with II and RsaI (including the P _L promoter, the DNA sequence of the protein N ′ lacking the C-terminal of the N protein, and the Shine-Dalgarno (SD) sequence region of the CII gene) RsaI break point, Eco
RI linker

[0051]

[Chemical 2] (Obtained from New England Biolabs) was added, the sticky end of the BgII breakpoint was made blunt with T4 DNA polymerase, and this DNA fragment was added with pBR 3
A plasmid (pS13) to which a DNA fragment ligated with T4 polymerase after blunting 22 with EcoRI was ligated (pS13)
Got

This plasmid (pS13) contains pBR3
The P _L promoter is located in the same direction as the transcription direction of the 22 tetracycline resistance gene (Tcr). Furthermore, this plasmid (pS13) was transformed with EcoRI and Sal.
The long fragment obtained by digestion with I was constructed into the plasmid pG1F4 (disclosed in JP-A-58-201995, Escherichia coli containing the plasmid was named SBMG 105, and was designated by MIC) as FERM BP-282. Foreign gene (human γ-type interferon gene Gl obtained by digesting the
The plasmid pS20 (see FIG. 5) is obtained by ligating the DNA fragment containing F).

One of the γ-hANP gene expression vectors p
S220 was carried out by using the plasmid M 13mp8-hANP 525 obtained in Example 2-2 with EcoRI and S as described below.
The fragment (about 510 bp) obtained by digestion with alI was
GlF obtained by digesting 20 with EcoRI and SalI
It was obtained by replacing the fragment containing That is, M13mp8-hANP525 DNA
EcoRI reaction solution containing 8 μg (100 mM Tris-HCl solution containing 50 mM NaCl and 5 mM MgCl ₂ , pH
7.3) After adding 15 units each of EcoRI and SalI to 70 μl and reacting at 37 ° C. for 1 hour, 1.0
% Agar gel electrophoresis separation was performed.

Next, the above-mentioned method (elec of Example 2-1)
An about 510 bp EcoRI-SalI DNA fragment containing the γ-hANP gene was extracted and purified by the Troelute method. On the other hand, 2 μg of the above pS20 plasmid was added to 50 μl of EcoRI reaction solution to obtain EcoRI and Sal.
10 units each of I was added, and the reaction was performed at 37 ° C for 1 hour
After 1.0% agar gel electrophoresis separation, the method described above (elec
EcoRI-SalID containing λP _L promoter and SD sequence of CII gene by trselute method)
An NA fragment (about 4.3 Kb) was obtained.

Then, an aqueous solution (3 μl) of the EcoRI-SalI DNA fragment containing the above-mentioned γ-hANP gene.
And EcoRI-SalI DNA of pS20 plasmid
In a solution of the fragment with 10 μl of an aqueous solution, 3 μl of a 10-fold concentration ligation solution (described in Example 2-1), 100 mM
DTT (dithiothreitol) 3 μl, 1 mM ATP
By adding 3 μl of an aqueous solution, 6 μl of distilled water and 2 units of T4 DNA ligase, and performing the reaction at 16 ° C. overnight,
One of the γ-hANP gene expression vectors, pS220, was constructed. For transformation of Escherichia coli N4830 strain (purchased from Pharmacia Japan) with pS220, 10 μl of the above reaction solution was treated with CaCl ₂ according to a conventional method.
To E. coli N4830 strain suspension containing 0 μl of 50 mM CaCl ₂ , the mixture was allowed to stand for 1 hour on ice, and then 2.5 ml of L was added.
-By adding broth and culturing at 32 ° C for 1.5 hours. Transformants were selected by isolating colonies (ampicillin resistant strains) growing on nutrient agar containing 50 μg / ml ampicillin.

Plasmid DNA was isolated from the obtained transformant by a conventional method and analyzed by restriction enzyme, and it was confirmed that the γ-hANP gene was inserted downstream of the λP _L promoter in the plasmid. . This transformant was designated as N4380 / pS220, and was used in the next experiment for production of γ-hANP.
In addition, the γ-hANP gene expression vector is the above pS22.
Other than 0, it was created as described below. That is, XbaI linkers [purchased from New England Biolabs: d (CTCTAGAG)] were added to DNA fragments of various sizes obtained by digesting plasmid pS20 with EcoRI and then hydrolyzing it with exonuclease Bal31.
Construct the plasmid pS20X ligated with
After digesting 0X with XbaI and SalI, pIN4GlF5
4 (disclosed in JP-A-60-24187) as Xb
The shorter DNA fragment (including the human γ-type interferon gene) obtained by cutting with aI and SalI is pS20.
X was inserted in place of the XbaI-SalI DNA fragment.

Among the plasmids thus constructed,
A plasmid having a high expression level of the γ-type interferon gene in the culture of Escherichia coli transformed with the plasmid (the method for measuring the expression level is disclosed in JP-A-60-24187) was designated as pS83-3. Next, as shown in FIG. 7, EcoRI-Sa of pS83-3.
Instead of the II fragment, the already obtained plasmid M13
Plasmid pS223-3 was obtained by inserting the EcoRI-SalI fragment of mp8-hANP525.
pS223-3 is an SD sequence of the Escherichia coli outer membrane protein (lpp) gene downstream of the λP _L promoter region (JP-A-60-
24187 (see FIG. 1)).
The transformant N4380 / pS223-3 obtained by transforming Escherichia coli N4380 with the plasmid is the above-mentioned N4.
It was subjected to an experiment for the production of γ-hANP with 380 / pS220.

Yet another γ-hANP gene expression vector pS224-3 is the plasmid pS83 described above.
Obtained by cleaving -3 with XbaI and EcoRI
Instead of the DNA fragment containing the SD sequence of the gene, a DNA fragment (AGGAGGT) having the SD sequence of the chemically synthesized bacteriophage MS2A protein gene having XbaI and EcoRI cleavage recognition sequences as sticky ends at both ends.
To obtain the plasmid pS84-3, and then, as shown in FIG. 7, E13 of M13mp8-hANP525 DNA.
The oRI-SalI fragment was added to EcoR of pS84-3 DNA.
Obtained by inserting the I-SalI fragment. pS
Escherichia coli N4380 / pS224-3 transformed with 224-3 in the same manner as above also had the aforementioned N4380 / pS.
Γ- with 220 and N4380 / pS223-3
It was subjected to an experiment for the expression of the hANP gene. 3. Production of γ-hANP by Transformant 3 transformants obtained in Example 2-3, namely N48
30 / pS220, N4830 / pS223-3, and N4830 / pS224-3 and the plasmid-free host strain N4830 in M9 minimal medium supplemented with 50 μg / ml of 19 natural amino acids except ampicillin and methionine. Cultivated at ℃, OD660 0.4
At that time, a part thereof was incubated at 42 ° C.

15 minutes and 30 minutes after transferring to 42 ° C., 2 μCi of ³⁵ S-methionine (manufactured by New England Nucleia Co., Ltd .: purchased from Japan Isotope Association) having a specific activity of 800 Ci / mmole was added to 0.3 ml of a culture solution. Plus 2 at the same temperature
After culturing for a minute, TCA (trichloroacetic acid) was added so that the final concentration was 10%, and the mixture was immediately cooled to 0 ° C. After 10 minutes, the precipitate was recovered by centrifugation, washed once with ethanol, and subjected to 15% SDS polyacrylamide gel electrophoresis separation according to a conventional method. The same treatment was performed on the sample that was continuously cultured at 32 ° C.

After the gel was dried, the band of the ³⁵ S-methionine-labeled protein was observed by radioautograph. As a result, as shown in FIG. 7, it was transformed with the plasmid into which the γ-hANP gene had been inserted. 3 transformants (N4830 / pS220, S4830 / p)
S223-3, and N4830 / pS224-3) were cultured at 42 ° C. in a control strain (N4830).
Also, a protein band corresponding to about 20 Kb, which was not found in the transformant cultured at 32 ° C., was observed.

Since γ-hANP purified from human atrium was electrophoresed at the same position in SDS polyacrylamide gel electrophoresis under the same conditions, the band of this protein was judged to be γ-hANP produced by the transformant. It was In addition, the fact that this protein is observed only in the culture at 42 ° C indicates that the γ-hANP gene is expressed under the control of the λP _L promoter.

As the standard protein kit for measuring the molecular weight in the above-mentioned electrophoresis, one manufactured by PL Biochemical was used.

[0063]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 843 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: cDNA Origin organism name: Human Direct origin: Plasmid phANP82 Sequence ACAGACGTAG GCCAAGAGAG GGGAACCAGA GAGGAACCAG AGGGGAGAGA 50 CAGAGCAGCA AGCAGTGGAT TGCTCCTTGA CGACGCCAGC ATG AGC TCC TTC 102 Met Ser Ser Phe TCC ACC ACC ACC GTG AGC TTC CTC CTT TTA CTG GCA TTC CAG CTC 147 Ser Thr Thr Thr Val Ser Phe Leu Leu Leu Leu Ala Phe GTA GGT CAG ACC AGA GCT AAT CCC ATG TAC AAT GCC GTG TCC AAC 192 Leu Gly Gln Thr Arg Ala Asn Pro Met Tyr Asn Ala Val Ser Asn 20 25 30 GCA GAC CTG ATG GAT TTC AAG AAT TTG CTG GAC CAT TTG GAA GAA 237 Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu 35 40 45 AAG ATG CCT TTA GAA GAT GAG GTC GTG CCC CCA CAA GTG CTC AGT 282 Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser 50 55 60 GAG CCG AAT GAA GAA GCG GGG GCT GCT CTC AGC CCC CTC CCT GAG 327 Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Se r Pro Leu Pro Glu 65 70 75 GTG CCT CCC TGG ACC GGG GAA GTC AGC CCA GCC CAG AGA GAT GGA 372 Val Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly 80 85 90 GGT GCC CTC GGG CGG GGC CCC TGG GAC TCC TCT GAT CGA TCT GCC 417 Gly Ala Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala 95 100 105 CTC CTA AAA AGC AAG CTG AGG GCG CTG CTC ACT GCC CCT CGG AGC 462 Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser 110 115 120 CTG CGG AGA TCC AGC TGC TTC GGG GGC AGG ATG GAC AGG ATT GGA 507 Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly 125 130 135 GCC CAG AGC GGA CTG GGC TGT AAC AGC TTC CGG TAC TGAAGATAAC 553 Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Try 140 145 150 AGCCAGGGAG GACAAGCAGG GCTGGGCCTA GGGACAGACT GCAAGAGGCT 603 CCTGTCCCCT GGGGTCTCTG CTGCATTTGT GTCATCTTGT TGCCATGGAG 653 TTGTGATCAT CCCATCTAAG CTGCAGCTTC CTGTCAACAC TTCTCACATC 703 TTATGCTAAC TGTAGATAAA GTGGTTTGAT GGTGACTTCC TCGCCTCTCC 753 CACCCCATGC ATTAAATTTT AAGGTAGAAC CTCACCTGTT ACTGAAAGTG 803 GTTTGAAAGT GAAT AAACTT CAGCACCATG GACAGAAGAC 843

[Brief description of drawings]

FIG. 1 represents the amino sequence of γ-hANP.

FIG. 2 represents an amino acid sequence consisting of a signal sequence and the amino acid sequence of γ-hANP.

FIG. 3 shows a nucleotide sequence encoding γ-hANP.

FIG. 4 shows a nucleotide sequence encoding a signal sequence and γ-hANP.

FIG. 5 shows a process of constructing a plasmid pS220 from a DNA fragment encoding γ-hANP and the plasmid pS20.

FIG. 6 is an electrophoretogram showing the expression of γ-hANP, which is a photograph as a drawing substitute.

FIG. 7 shows a DNA fragment encoding γ-hANP and plasmids pS83-3 and pS84-3,
Plasmids pS223-3 and pS224-3, respectively
Represents the process of creating.

FIG. 8 shows the nucleotide sequence of a probe used for selection of a DNA fragment encoding γ-hANP.

FIG. 9 shows the strategy used for determining the nucleotide sequences in phANP1 and phANP82.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI technical display location C12R 1:19) (72) Inventor Shoji Tanaka 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Prefecture No. Suntory Co., Ltd. Biomedical Research Institute (72) Inventor Hiro Nakazato 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun Osaka Prefecture Suntory Co., Ltd. Biomedical Research Institute (72) Yasunori Tawaraki Shimamoto-cho, Mishima-gun Osaka Prefecture Wakayamadai 1-1-1 Suntory Co., Ltd. Biomedical Research Institute (72) Inventor Toshiyuki Matsuo 5-15-141 Onohara Higashi, Minoh City, Osaka Prefecture (56) Reference JP-A-60-215698 (JP, 60, 215698) A) BIOCHEMICAL AND BI OPHYSICAL RESEARCH COMUNICATION, 1984-118! P. 131-139

Claims (2)

[Claims] 1. Amino acid 26 (A in SEQ ID NO: 1
a human diuretic effect having or containing an amino acid sequence from sn) to amino acid 151 (Tyr)
A method for producing a polypeptide having the method, comprising producing the polypeptide by culturing a bacterium transformed with an expression vector containing a DNA encoding the polypeptide, and then collecting the polypeptide. And how to. 2. The polypeptide is amino acid 1 (Met) to amino acid 151 (Tyr) in SEQ ID NO: 1 .
The method of claim 1, which is a polypeptide having an amino acid sequence up to.