JPS626689A - Synthetic gene - Google Patents

Abstract

NEW MATERIAL:A synthetic gene capable of coding an amino acid sequence of an alpha-human atrial-derived sodium excreting polypeptide expressed by the formula. USE:Formation of a recombinant vector or transformant and production of a diuretic and hypotensive agent using said recombinant vector or transformant. PREPARATION:The respective oligonucleotides (AH2-AH17) of blocks I''' and II''', e.g. Figure 15, are phosphorylated with a T4 polynucleotide kinase in a ligation buffer, and T4DNA ligase and ATP are added and reacted therewith. Unphosphorylated oligonucleotides (AH1 and AH18), T4DNA ligase and ATP are then mixed with the resultant mixture and reacted therewith to afford the final ligation product, which is purified by 2-16% gradient PAGE to give the aimed synthetic gene, capable of coding an amino acid sequence of alpha-hANP and having linker DNA and 134 b.p., e.g. Fig. 5.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a novel synthetic gene. More specifically, α-human atrial natriuretic polypeptide (α-human atrial natriure
tic polypeptide, hereinafter referred to as “α-hANP
”) and a synthetic gene encoding a protected peptide-fused α-hANP, a recombinant vector and transformant containing the gene, and α-hA produced by the transformant.
The present invention relates to a method for producing NP.

Laparotomy A α-hANP, which is to be solved by the prior art and this invention, has a diuretic effect, a natriuresis-increasing effect,
It is a known polypeptide that has antihypertensive effects, is useful as a diuretic antihypertensive agent, and is a known polypeptide that has antihypertensive effects.
5er-Phe-Ar (r)
(See Biochemical and Biophysical Research Communications Vol. 118, p. 131 (1984)).

A method for producing this α-hANP with good yield has been desired.

The present inventors have created a new method for producing α-hANP by recombinant DNA technology using an expression vector containing a synthetic gene encoding the amino acid sequence (I) of α-hANP. According to this method, α-hANP can be obtained in high yield.

In this invention, (1) a microorganism transformed with an expression vector containing a synthetic gene encoding the amino acid sequence of protected peptide-fused α-hANP is cultured in a medium;
2) Protected peptide-fused α-hANP from the resulting culture
and (3) removing the protected peptide portion of the protected peptide-fused α-hANP to produce α-hANP.

The details of the manufacturing method described in E will be explained in more detail below.

The microorganism to be transformed is a host cell, including bacteria,
Mention may be made of fungi, human and animal cultured cells and plant cultured cells. Preferable examples of microorganisms include bacteria, especially ≧, Shrimp Book 101 (ATCC 33694)
, Dan, Manufacture 294 (ATCC31446), E-, c
oli X 1776 (AlCC31537) etc. (7
) Engineering'/ Bacterial strains belonging to the genus Erichia can be mentioned.

Expression vectors usually consist of DNA having at least a promoter operator region, an initiation gene, a synthetic protected peptide gene, a synthetic α-hANP gene, a stop codon, and a replicable unit.

The promoter-operator region contains a promoter, an operator, and a Shine-Dalgarno (SD) sequence, such as AAGG. The distance between the SD sequence and the start codon is preferably 8 to 12 base pairs (b, p,), and in the most preferred example as shown in the Examples below, the distance between the SD sequence and the start codon is 11 b. , p
, is. Examples of promoter/operator regions include, in addition to synthetic promoter/operator regions, the lactose operon, PL-promoter, trp
- Commonly used promoter/operator regions such as promoters can be mentioned. A preferred example of the promoter/operator region is the synthetic arp promoter newly synthesized by the present inventors! , ■, and ■, whose DNA sequences are shown in Figures 1, 2, and 3, respectively.
In the production method of the present invention shown in the figure, one to three promoter/operator regions may be used for each expression vector.

A preferred start codon is the methionine codon (ATG
) can be mentioned.

Protective peptide genes include genes that encode the amino acid sequence of a peptide or protein that can form a fusion protein with α-hANP and that have a DNA sequence that suppresses undesirable degradation of the fusion protein in host cells or cultures. can be mentioned.

A preferred example thereof is a ``peptide Cd gene'' connected to the ``LH protein gene'' (hereinafter, the peptide Cd gene connected to the ``LH protein gene'' is referred to as the ``peptide CLa gene''), and its DNA sequence is As shown in the figure.

The DNA sequence of the α-hANP gene is designed from the amino acid sequence of α-hANP according to a number of non-obvious criteria. An example of a preferred DNA sequence of the α-hANP gene is shown in FIG. In the examples described later, α-h
A DNA sequence encoding the amino acid lysine was inserted between the ANP gene and the protected peptide gene.

In this way, the junction of the fusion protein can be cleaved with Achromobacter protease (2).

Examples of the termination codon include commonly used termination codons such as TAG and TGA.

A replicable unit is a DNA sequence that is capable of replicating the entire DNA sequence belonging to it in a host cell, and includes natural plasmids, artificially modified plasmids such as DNA fragments prepared from natural plasmids, and synthetic plasmids. can be mentioned. Furthermore, a preferable example of the plasmid is plasmid pBR322 or an artificially modified version thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme). The replicable unit may include natural or synthetic terminators (eg, synthetic fd phage terminators, etc.).

When synthesizing a promoter/operator region, initiation codon, protected peptide gene, α-hANP gene, termination codon, terminator, etc., the synthesis method can be performed by a known method commonly used for producing polynucleotides. .

The promoter operator gene, start codon, protected peptide gene, α-hANP gene and stop codon can be used as appropriate NA fragments (e.g. linker-1), if desired.
A suitable replicable unit (plasmid) is prepared using conventional methods (e.g., digestion with restriction enzymes, phosphorylation using T4 polynucleotide kinase, ligation using T4 DNA ligase, etc.) using other restriction enzyme action sites, etc. An expression vector can be obtained by ligating continuously and circularly.

Expression vectors can be inserted into microorganisms (host cells) by conventional methods. Insertions can be made, for example, by transformation, micro-
Transformants can be obtained by conventional methods such as injection.

To produce α-hANP in the production method of this invention,
This is carried out by culturing the transformant containing the expression vector thus obtained in a medium.

The medium may be any medium that allows the transformant to grow, such as carbon sources such as glucose, glycerin, mannitol, fructose, and lactose, and ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypeptone, and batatotryptone. , Beefex, etc., and if desired, inorganic salts such as sodium or potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride, etc., such as vitamin B1, etc. Other ingredients such as vitamins, antibiotics such as ampicillin may also be added to the medium.

The transformant is usually cultured at pl (5.5 to 8.5 (preferably pH 7 to 7.5) and 18 to 40°C (preferably 25 to 38°C) for 5 to 50 hours.

Since the protected peptide-fused α-hANP produced in this way normally exists in cultured transformant cells, the cells are collected by filtration or centrifugation, and the cell walls and/or cell membranes are removed by conventional methods (e.g., ultrasonication). and/or treatment with lysozyme, etc.) to obtain destroyed cells. From this destroyed material, conventional methods commonly used for the purification and isolation of natural or synthetic proteins (for example, dissolving the protein with an appropriate solvent such as 8M JIR hydrogen aqueous solution, 6M guanidine, etc.)
peptide fusion α-hAN protected by dialysis, gel filtration, column chromatography, high performance liquid chromatography, etc.)
P can be purified and isolated.

α-hANP can be prepared by cleaving protected peptide-fused α-hANP by treatment with an appropriate protease (e.g., Achromobacter protease I (API), etc.) or by chemical methods (e.g., treatment with cyanogen bromide). can. When the C-terminus of the protected peptide is lysine, treatment with API is preferably used. API
is a known enzyme [Biochimica Et. Biofig. Acta, 660.51 (1981)], but the recombinant D
It is not known that fusion proteins prepared by NA technology can be preferably cleaved by API treatment.

This method can be suitably used to cleave a fusion protein consisting of a peptide that has lysine between the protected peptide and the target peptide that does not have lysine in its molecule.

Cleavage of the fusion protein may be carried out at pH 5 to 10 and at 20 to 40°C (preferably 35 to 40°C) for 2 to 15 hours in an aqueous solution such as a buffer solution or an aqueous urea solution.

In the Examples below, the fusion protein was first incubated in a pH 5 buffer containing 8M urea and then in a pH 5 buffer containing 4M urea.
9 buffer with API. Under these conditions, the fusion protein is cleaved at the lysine site, producing α
- hANP is spontaneously reconstituted.

α-hANP thus produced is purified and isolated from the obtained culture by the above-mentioned conventional method.

The figures attached to this specification will be explained below.

In some of the figures, the oligonucleotide is
Indicated by the symbol □ □ or □ (in the symbol, . means the 5'-end phosphorylated by T4 polynucleotide kinase), the blocked oligonucleotides are -, m, m, -mushiichi, - It is indicated by the symbol 1 or 1^- (in the symbol Δ means the ligated position).

Unless otherwise specified, in this specification, DNA
A, G, C, and T in the sequence mean each of the 4 formulas: 5'-end A%G, C%T mean each of the 4 formulas: 3'-terminus A, G
, C, and T each mean 4 formulas:

In the following examples, Ap, Gp, Cp, Tp
means each of the four formulas: 3'-terminal AOH, GOH, COH%TOH
means each of the four formulas: 1,5' terminal HOAp, HOGp, HOC
p, HOTp mean each of the following four formulas: % formula % formula: DMTr is dimethoxytrityl, C
E is cyanethyl.

Mono(or di- or trimer) oligonucleotides can be prepared using conventional methods such as the method of Hirose et al. [Proteins, Nucleic Acids, Enzymes 2
5.225 (1980)] and couplings can be prepared, for example, by the phosphotriester method [Newcratic Acid Research, 1691 (1980)] on cellulose or polystyrene polymers.
1), New York Ashinod Research,
1755 (1982).

The following examples are intended to illustrate the invention and are not intended to limit it.

In the examples, the enzymes used (e.g., restriction enzymes) are commercially available, and the conditions for using the enzymes will be obvious to those skilled in the art, for example, by referring to the instructions attached to the commercially available enzymes. be.

Furthermore, in the examples, 1-boristyrene borimaya means aminomethylated polystyrene HCl, divinylbenzene content 1%, 100-200 mesh (sold by Peptide Institute, Inc.).

Example I HOCpTpGpCpGpTpApGpApTpCpC
Synthesis of pTpCpTOH (A) 17): (1) Synthesis of DMTrOTpoC"2polo-succinyl polystyrene polymer 1) Preparation of HOTO-succinyl polystyrene polymer: DMTrO-IO-succinyl polystyrene polymer (51,8 mg, 10 .37μmol) [Newcratsik Acid Research 10°17
55 (1982)] is added with a 5% dichloroacetic acid # (DCA) solution in dichloromethane (2 m). After standing for 1 minute, the mixture is filtered through a glass filter with a stream of nitrogen. Repeat the DCA process two or more times. Polymer dichlorometh'7 (2mQX3)
, methanol (2mux3) and pyridine (2mQx
3) and dried in a nitrogen stream to obtain a polymer-applied product ①.

N) Preparation of DMTrOTpoCB2po-: DMTrOTpoC"po-CE (32,
4 mg, 8.12 μmol) was added to a triethylamine-acetonitrile mixture (1: lv/v,
5mQ). The phosphodiester dimer thus obtained (DMTrOTpoC""po-) is dried and the water is separated off as an azeotrope with pyridine (2mQX2).

i) Coupling: dimer (DMTrOTpoC"po-) and mesitylenesulfonyl nitrothiazolide (MSNT) (80 m
g) in pyridine (o, smσ). The solution is added to the reaction syringe along with Polymer Adduct 1 and the mixture is shaken for 1 hour at room temperature. The reaction mixture was filtered through a glass filter with a stream of nitrogen and treated with pyridine (2 mn x 3).
Wash with water to obtain polymer adduct (■).

iv) Acetylation of unreacted 5'-hydroxy groups: Pyridine (0,9
mQ ) and acetic anhydride (0,1 mA) are added and the mixture is shaken for 15 min. The reaction solution was filtered through a glass filter, and the resulting polymer was mixed with pyridine (2mQx3).
, methanol (211111X 3 ) and dichloromethane (2mux3) and dried with a stream of nitrogen. This polymer adduct (DMTrOTpoC'poT
o-succinyl polystyrene polymer) was used in the next force 7 bling step.

(2) DMTrOTpoC""poC""porpo
Synthesis of c''poro-succinyl polystyrene polymer: Under the same conditions as <1) above, DMTrOIpoC''po
TO-succinyl polystyrene polymerate DMTrOT
D from poC"poC"poCE (44.9mg)
MIrOTpoCB"poc""poTp.

cB zpoτO-succinyl polystyrene polymer is synthesized.

(3) DMTrOA87'poGiBpoA87'p
Synthesis of oTpocB2poCB2poTpoC"poro-succinyl polystyrene polymer: Under the same conditions as in (1) above, DMTrOrpoC""p
oc”poTpoCB2poTo-succinyl polystyrene polymer and BZ iB B2 DMTrOA poG poA poCE (48
, 5mg, the opening τrOA'poGlBpoA""p
oTpocB"poc"poTpocB2poTo-succinyl polystyrene polymer is synthesized.

(4) DMTrOCB2poGiBpoTpoAB2
poGiBpoAB"po 工pocBzpoC"poT
Synthesis of poCB"poTO-succinyl polystyrene polymer: Under the same conditions as in (1) above, DMTrOA poG
poAporpoc""poC""poTpoC""
poIo-succinyl polystyrene polymer and DMTr
OCB2poGiBpoTpocE (45,1mg
), DMTrOC"poG'LBpo?poA""
poGiBpoA””poffp.

CB2poC"poTpocB2'poTo-succinyl polystyrene polymer is synthesized.

(5) DMTrOCB2poTpoGiBpoCB2
poGiBpoTpoA"2poGiBpoA"por
Synthesis of poc""poC""poTpocB2poTo-succinyl polymer: Under the same conditions as above (1), DMTrOC"2poGi
Bporp.

AB2poGiBpoA”po?poCB2poC”2
polpoC""poTO-succinyl polystyrene polymer and DMTrOC""poTp.

From GlBpoCE (45, 1 mg), DMTrOC
B2poTpoGiBp.

C""poG iBp, JTpoA"poG'"poA
””poTpoCB”poC””poIp.

C”poTo-succinyl polystyrene polymer (60
mg) is synthesized. In this final step, the unreacted 5
The '-hydroxy group does not need to be protected with an acetyl group.

(6) )lOCpTpGpCpGpTpApGpAp
Synthesis of TpCpCpTpCpTOH: DMTrOCB2poTpoGiBpoCB2poGi
BpoTpoAB2poGiBp.

A"poTpoC"poCB2porpoc""pol
o-succinyl polystyrene polymer (60 mg),
1, IM N, N, N', N' - in a sealed tube at 37°C.
Treat with tetramethyleneguanidium pyridine-2-aldoximate [in dioxane-water (1:1 v/v, 1 mc)] for 20 hours. 28% Cw/w in the reaction mixture
) Add aqueous ammonia (12ffl11) and heat the mixture at 60°C for 5 hours. The solid polymer was filtered off and water (
10ml). Evaporate solution a and washing solution to dryness,
The residue is treated with 80% aqueous acetic acid (25 mQ) for 15 minutes at room temperature. After removing the solvent, the residue was reduced to 0. Dissolve in IMN acid triethylammonium buffer (pH 7, 5, 25 mB) and wash with diethyl ether (3 x 25 mQ). The aqueous layer is evaporated to dryness and the residue is dissolved in 0.1 M triethylammonium carbonate buffer (pH 7, 5, 2 mQ). When dissolved in the solution, the crude HOCpTpGpCpGpTpApG
pAplpCpCpTpCpTOH is obtained.

(7) HOCpTpGpCpGpTpApGpApT
Purification of pCpCpTpCpTOH 1) Initial purification of the crude product was carried out using Biogel P2 (Bio-Rad) G24x2.
6 cm ID) column chromatography. Both fractions corresponding to the first elution peak (50mM ammonium acetate containing 0,1mM MEDIA, flow rate: 1rnQ/min) are collected and lyophilized to obtain the first purified product.

■) The second purification of the first purified product was performed by high performance liquid chromatography (HPLC) using CDR-10 (Mitsubishi Kasei) (25 cm x 4.6 mm ID) from 1M ammonium acetate-10% (V/V) aqueous ethanol. 4
．． Linear gradient from 5M ammonium acetate to 10% (V/V) aqueous ethanol (80 minutes, flow rate: 1 mQ/min, 6
0°C) to obtain the second purified product.

11) The third purification of the second purified product is performed by reverse phase HPLC.
[Rp-18-5a (X 77 ) (Merck), 1
5cm x 4mm ID ] by 0.1M vinegar 0.1M ammonium acetate 1M ammonium acetate - 15% (V/V)
Linear gradient to aqueous acetonitrile (40 minutes, 1.
5 mQ/min, room temperature) to obtain the final purified product.

(HOCpTpGpCpGpTpApGpApTpCp
CpTpCpTOH) (8) Oligonucleotide (HO
CpTpGpCpGplpApGpApTpCpCpT
Analysis of pCl)TOH) 1) Digestion with phosphogesterase HOCpTpGpCpGpTpApGpApTpCpC
pTpCprOH (10 to 5 countries), o, 2M
MgC1z (204), 0.2M Tris-)I
C1(p)Is, 5') (20 bound) and 0.1
A mixture of mM EDTA (144,9%) is treated with phosphogesterase (10 units, lOS) for 20 minutes at 37°C and then heated at 100°C for 2 hours.

i) HPLC Analysis Oligonucleotides in the reaction mixture were analyzed by HPLC [CDR
-10 (Mitsubishi Kasei), 25cm x 4.6mm ID by water~2. Linear gradient of OM ammonium acetate (pH 3,4) (40 min, flow rate: 1.5 mA/min, 60 °C
) to analyze. Determine its nucleotide composition from each peak area by comparing with the area of a standard.

Calculated values: pcOH4,000, pAOH2,000,
pTOH5,000゜pGOH3,000 Actual Hong 1 value: pcOR3,770, pAOH2,026
, pTOH5,237゜pGOH2,968゜Synthesis of main oligonucleotide: The following oligonucleotide is prepared in the same manner as described in Example 1.

(1) HOApGpCpTpTpGpApApGp
IprpGpApGpCpApTpGOH (All) (2) HOApApTpTpCpApTpGpCp
TpCpApApCplpTpCpAOl ((Al1) (3) HOApApTpTpCpGpGpTpA
pTpGpGpGpCOH (Al1) (4) HOT
plpCpApCpCpGpCpCpCpApTpAp
CpCpGOH(Al1> (5) HOGpGpTpGpApApGpCpTp
ApApApTpCpTOH (Al1) (6) HO
CpGpCpApGpApGpApTpTpTI)Ap
GpCOH(Al1) (7) )IOApApGpC
pApApGpApGpGpApTpCpTpAOH (
Al1) (8) HOTpGpCpTpTpTpGp
GpTpGpGpCpCpGpIOH (Al1) 3°ゝ゛°“pCpCpAp”′”pCpGpGpCpCp”
pCpCp””(AHIO)3”O) HOAp”pG
pGpApCpCpGpCpAp"pCpGpGpT°
'(AHII) (11) HOTpGpApGpCpA
pCpCpGpApTpGpCpGpGOH (All2
) (13) HOCpApGpCpCpCpApGpAp
CpCpGpGpApCOH (All4) 3"ゝ)lOGpGpcp"'°"pApApCp"'
°“′”“pcOH,,,□1.>(16) HOCp
GpTpTpApCpTpGpApIpApGOH (A
ll7) (17) HOGpApIpCpCpTpAp
lpCpApGOH(All8)]d(Synthesis of 'II oligonucleotide-: The following oligonucleotide is prepared in the same manner as described in Example 1.

(1) HOApApTpTpTpGpCpCpGp
ApCpAOH(A)(2) HOCpGpTpTp
ApTpGpApTpGpTpCpGpGpCpAOH
(B) (3) HOTpCpApTpApApCp
GpGpTpIpCpTpGpGpCOH (C) (4)
HOGpApApTpApIpTp'rpGpCp
CpApGpApApCOH (D) (5) HOAp
ApApTpAprpTpCprpGpApApApr
pGpAOH<E) (6) HOTpCpApAp
CpApGpCpTpCpApTplpTpCpAOH
(F) (7) HOGpCpTpGpTpTpGpA
pCpApApTpIpApAprOH (G) (8)
HOGpTpTpCpGpApTpGpApTpTp
ApApTpTpGOH (H) (9) )IOCpA
pTpCpGpApApCpTpApGpTpIpAp
ApCOH (I) (10) HOGpCpGpTpAp
CpTpApGpTpTpApApCpIpAOH (J
) (11) HOTpApGplpApCpGpCpA
pApGpTpTpCpApCOH (K) (12) H
OCplpTpIpTpTpApCpGpTpGpAp
ApCpTpIOH (L) (13) HOGpTpA
pApApApApGpGpGpTpApTOH (M'
) (14) HOCpGpApTpApCpCOH (
N') (15) HOGpTpApApApApApG
pGpGpTpApTpCpGOH (M) (16) H
OApApTpTpCpGpApTpApCpCOH (
N) (17) HOApApTpTpCpAprpGp
GpCpTOH (SA) (19) )IOTpTpTp
GpGpApApGpApCplpTpIOH (SC)
(21) HOTprpApCpApApCpCpA
pGpCpCpApTpGOH (SE) (22) HO
CpCpApApApApGpApApGpTpTpC
OH(SF)<23> )lOCpGpApApGpT
pGpApApApGpTpCpTpTO)I (SG
) (24) HOGpApTpCpCpTpApTp
Synthesis of oligonucleotides: The following oligonucleotides are produced in the same manner as described in Example 1.

(1) HOApApCpTpApGpTpApCp
GpCOH(Npl) (5) HOGpGpTpAp
TpCpGpApTpApApApAp'rpGOH (
Np3) (7) HOIpTpCpTpApCpTp
TpCpApApCpApApAO)l (Cdl)(
8) HOGpGpTpCpGpGpTpTpTpG
pTplpGpApAOH(Cd2) (9) HOC
pCpGpApCpCpGpGpCpTpAprpGO
H(Cd3)(10) HOGpCpTpGpGpAp
GpCpCpApTpApGpCpCOH (G2) (1
1) HOGpCpTpCpCpApGpCpTpCp
TpCpGpTpCOH (Hl) (12) HOCpG
pGpTpGpCpGpCpGpApCpGpApGp
AOH()+2)(13) HOGpCpGpCpAp
CpCpGpCpApGpApCpTpGOH(It)
(14) HOGpAprpApCpCpApGpTp
CpTpGOH (Cd4) (15) HOGpTpAp
TpCpGprpApGpApCpGOH (Cd5) (
16) HOApCpCpCpTpCpGpTpCpT
pApCOH(Cd6) (17) HOApGpGpG
prpGpGpCpGpAp'rpGOH (Cd7) (
18) HOApApTprpCpApTpCpGpC
Construction and cloning of the pCOH(Cd8)W promiscuously synthesized trp promoter ■ gene (shown in Figure 6.7): The trp promoter ■ gene was constructed using the same method as the jj method described in Example 7 (shown in Figure 6). To construct.

The synthetic gene was ligated with the EcoRI-BamHI fragment of pBR322 (commercially available from Takara Shuzo, NEB, etc.), and then immediately placed in the device HBIO1 (AlCC3
3694) with the ligation product. R
Plasmids obtained from Amp and 5Tet transformants were digested with HpaI and identified by polyacrylamide gel electrophoresis (PAGE), followed by digestion with BamHI and PAGE to identify 90b, p, The band was confirmed. Furthermore, EcoRI-BamHI
The fragments of 56b, p, resulting from the digestion were confirmed by PAGE and comparison with size markers. This plasmid was named pTrpEB7 and was used to construct an expression vector.

trp promoter vector (pBR322trp)
Construction and cloning of the niblasmid pBR322 (9,4) (shown in Figure 8) with EcoRI and BamH.
Digest with I restriction endonuclease. The reaction was stopped by heating at 65° C. for 5 minutes, and the fragments were separated by 0.8% agarose gel electrophoresis to obtain a small fragment (500 ng) of 375b,p. On the other hand, plasmid pTr
pEB7 (to < ) with EC0RI and BamHI
4094b, followed by preparative gel electrophoresis.
, 9. A large fragment (5() is obtained. pTrpEB
EcoRI-BamHI fragment of 7 (4094b, p,
, 200 < ) of pBR322 with EcoRI-Bam
Ligation buffer [50mM Tris-HCI (pH 7,
6), 10mM MgCl2.20mM DTT, 1
mM ATP, 1mM spermidine, 50 g/
Ligate overnight at 15°C in mlBSA (20 mL). With this ligation mixture, the apparatus HB
The IOI was calculated using Kushiner's method [T, 1 (6nia
tis et al., Molecular Cloning, p252(1
982), transformed by Cold Spring Harbor Laboratory and treated with tetracycline (25x
/mQ) to obtain transformants resistant to tetracycline. The plasmid pBR322trp isolated from the transformant was incubated with EcoRI-Ba.
Digested with mHI (375b,p,, 4094b,p,) and HpaI (4469b,p,) and 7.5
The trp promoter gene was confirmed by %PAGE and 0.8% agarose gel electrophoresis.

Synthetic trp promoter ■Construction of gene (shown in Figure 9) Each orinucleotide (B-M') (0.2 nmol each) of Ni blocks I', n' and ■' (0.2 nmol each) was added to T4 polynucleotide. Phosphorylate with Kinase (BRL, 2.5 units) in ligation buffer (704) at 37°C for 1 hour. T4DN in the reaction mixture of each block.
A ligase (300 units) and 20nn MATP (2
4) and incubate the mixture at 15°C for 30 minutes, then heat at 65°C for 10 minutes to stop the reaction.

Each of these blocks (1', I' and III'
) reaction mixtures were combined and treated with T4 DNA ligase (360
units) and non-phosphorylated oligonucleotides (A, N
') to mix. After incubating the mixture for 1 hour at 15 °C, the final ligation product was 2-16%
Gradient polyacrylamide gel electrophoresis CPAGE
) to obtain the synthetic trT) promoter ■ gene of 106b,p.

X odor counterfeit 1 double trp promoter vector (p322dtrp
Construction and cloning of the niblasmid pBR322trp (shown in Figure 10) with EcoRI and C1
Digestion with aI followed by preparative agarose gel electrophoresis yields the large fragment of 4446b,p. This fragment (
4446b, p, ) was ligated with the trp promoter (106b, p, ) obtained in Example 7 in the presence of T4 DNA. In this ligation mixture E,
, coliHBlol is transformed to obtain transformants resistant to ampicillin and tetracycline. Plasmid p322dtrps obtained from this transformant
was confirmed by restriction endonuclease analysis. C
1aI-BamHI (325b, p, ), HpaI
(107b, p,) and Aat II-C1aI
(287b, p.).

Example 9 Construction of a peptide Cd gene having a portion of the DNA fragment of the synthetic trp promoter ) (Npl-Cd8, shown in Example 4)
using T4 polynucleotide kinase (2,5 units) in ligation buffer (60s11).
Phosphorylate for 1 hour at 7°C. Add T4 DNA ligase (360 units) and 20 m
MATP (2A) is added and the mixture is incubated at 15 °C for 1 h. Combine the reaction mixtures of each block, add T4 DNA ligase (360 units) and 20m
Incubate with MAYP (2It) at 15°C overnight, followed by heating at 80°C for 10 minutes.

Add 5QOmM sodium chloride aqueous solution (zofi
) and EcoRI (20 units) are added. 37°C
After incubation for 2 hours, the final ligation product is purified by 15% PAGE to obtain the peptide Cd gene with part of the DNA fragment of the synthetic trp promoter. This DNA sequence is shown in FIG.

Example 10 Construction and cloning of plasmid pCd7 (first
3) plasmid p7trp (4544b, p,) (shown in Figure 3)
E. coli F-9 containing this plasmid has been deposited as FERM BP-905 at the Microelectronic Research Institute. The large fragment (451
0b, 9. ). Synthesis t obtained in Example 9
A peptide Cd gene containing a portion of the DNA fragment of the rp promoter (■) is ligated in the presence of T4 DNA ligase. Using this ligation mixture,
Transform liHBlol. The plasmid (pCdy) obtained from the Amp transformant was confirmed by restriction endonuclease analysis (1aI-BamHI
(543b, p,), C1aI-HindIll (2
73b, p, ), C1aI-EcoRI (93b,
9. ) and Aat If -C1aI (180b
, I),).

Construction and cloning of plasmid pCd7trpsd (shown in Figure 14) A small fragment (543b, p, ) was obtained by digesting the plasmid pcct7 with C1aI and BamHI, which was then digested with C1aI of p322dtrpS (Example 7). -Ba
Ligate with the mHI fragment (4223b, p,) in the presence of T4 DNA ligase. This ligation mixture is transformed into ii. E. coli HBloI.

A plasmid (pcd) obtained from a transformant of RAmp
7 erpsd) was confirmed by restriction endonuclease analysis. HpaI-BamHI (1075,575b
, p, ), C1aI-BamHI (543b, p,
), PstI-EcoRI (1057b, p, ),
EcoRI-BamHI (450b, p, ), Hi
ndl[[-BamHI (270b, p, ) and C1aI-HindI [[(273b, p, ).

Example 12 Preparation of α-hANP gene with linker DNA (first
5) Niblock I″′ and ■″′ oligonucleotides (shown in Figure 5)
Al1-AHI7) (0.2 mol each) were treated with T4 polynucleotide kinase (2.5 units) in Regen John Huffer (704) for 1 hour at 37°C.
Phosphorylate. τ4D in the reaction mixture of each block
NA ligase (300 units) and 20mM AYP (
2p) and incubate the mixture for 30 minutes at 15°C. The reaction is stopped by heating at 65°C for 10 minutes. The reaction i1 compounds of the two blocks CI"' and I[') were combined and combined with nonphosphorylated oligonucleotides (AHI, AHlg) in the presence of T4 DNA ligase (300 units) and 20 mM AYP (24).
Mix with. After incubating the mixture for 1 hour at 15°C, the final ligation mixture was purified by 2-16% gradient PAGE and the 134b with linker-DNA
, p (shown in FIG. 5).

Example 13 Construction and cloning of α-hANP expression vector pcLaHtrpsd (shown in Figure 16) Niblasmid p(d 7 trpsd was digested with 1indn [and B, amHI to generate large fragments (4743b, p,
') is obtained, and this is ligated with the α-hANP gene (134b, p, ) having a linker DNA in the presence of T4 DNA ligase. This ligation mixture is used to transform HBIOI to obtain transformants. Plasmid (pcLaHtrpsd) obtained from a transformant of 2A knockout (≧, coliHl)
[It contains the CLaH protein (peptide CLa fusion α-hANP protein) gene, whose DNA sequence is shown in FIG. 17, and was confirmed by restriction endonuclease analysis. Aatll-C1aI (287b, p,), C1
aI-BamHI (407b, p, ), C1aI-
EcoRI (93,198b, p, ), EcoRI
-BamHI (116,198b, p, ), Hin
dll[-BamHI (134b, p, ) and Hp
aI-BamHI (107,439b,p,).

Expression of the gene that feeds the shakugogodan peptide CLa-fused α-hANP (CLaH protein): Ampicillin 50</mfJ-containing Lpros (29mM
), the expression vector (plasmid pcL
A culture of E. coliHl containing aHtrpsd) was grown in M-9 medium (containing 0.2% glucose, 0.5% casamino acids (casein hydrolyzate), 50t4/IQ vitamin B1 and 254/rnQa〉picillin). 4oo
E. coli is then cultured at 37°C. A600 (absorbance at 600 nm) of the culture solution is 0.
5, β-indole acrylic acid (2 mg/
ml ztanol, 2 mQ) is added and the cells are incubated for 3 hours (final A 600-1.85).

Next, the bacterial cells were centrifuged (6000 rpm).

4° C. for 5 minutes).

Isolation and purification of α-hANP: (1) Peptide CLa fusion a-hANP (CLaH
Separation and purification of protein): The fa-hydrated bacterial cell paste obtained from the culture solution (600 mQ) prepared in Example 14 was diluted with 10 mM PBS-EDTA.
(pH 7.4) [NaCl in 1 liter (8.0 g,
), KCI (o, zg ), Na2HPO4-12H
20 (2,9g'), KH2PO4 (0,2g),
EDTA (3,73 g) suspended in 18aI and cells disrupted by superg01 treatment at 0"C. 15.00 Orp.
Collect the pellet by centrifuging for 20 min (4'C) at
This was mixed with 6M guanidine-HCl, 10mM PBS-E.
Suspend in 13ml of a mixture of DTA and 2mM β-mercaptoethanol and sonicate the suspension at 0°C.

This suspension was heated at 15,000 rpm for 20 minutes (4°C).
After centrifugation, the supernatant was added to 10+nM PBS-E containing p-nitrophenylmethylsulfonyl fluoride (PMSF').
Dialyze against DTA solution overnight at 4°C. The dialyzed fraction was centrifuged (15,000 rpm, 4°C).

After 20 min (15 fl), the pellet was treated with 6M guanidine.
Dissolve in 100 mM Tris-HCI buffer (1)H8,O) (8 mm) containing HCl, 10 mM EDTA and 100 mM dithiothreitol and leave the solution overnight. This solution is dialyzed twice using 1M acetic acid (o, 5 liters) containing 10mM 2-mercaptoethanol and adjusted to pH 8.0 with trisaminomethane. The resulting precipitate (fusion protein; 15.2 mg) was centrifuged (3,
00Orpm for 10 minutes) and washed with 10mM sodium acetate buffer (pH 5.0).

(2) Achromobacter protease I (AP I
) Dewt of peptide CLa from peptide CLa-fused α-hANP: The fusion protein obtained above was suspended in 10 mM sodium acetate buffer (pH 5,0) (30 mM) containing 8 M urea, and the suspension was mixed with Achromobacter -・Protease I (A
PI) (0,25 units) (Wako Tsumugi Pharmaceutical), 37
Incubate for 2 hours at °C. The reaction mixture was diluted with distilled water (3 omQ) and adjusted to pH 9 with trisaminomethane.
,0, then add more API (0,25 units) and incubate for 2 hours at 37°C. 1 of the reaction solution
0mM sodium phosphate buffer (pH 7,0) (12
dilute with 0 mQ) and adjust the pH to 7 with acetic acid. This solution was equilibrated with a 10 mM sodium phosphate buffer (pH 7.0) on a Sp-Sephadex C-25 column (15
111Q). The column was washed with the same buffer and
Eluted with 10 mM sodium phosphate buffer (pH 8.0) containing 0.5 M' sodium chloride aqueous solution, and fractions containing partially purified α-hANP (0.4 mg) were collected.

3> High performance liquid chromatography (HPLC): the above (
The pooled fractions obtained in the same manner as in 2) were concentrated under reduced pressure, dialyzed against water (3 oomu), and subjected to reverse phase HPLC.
to obtain pure α-bANP (0.3 mg).

HPLC conditions (preparation) Beckman Ultra Bore for two-hand column preparation (φ1010
X250) Flow rate: 2.5+119/min elution, 0. OIM Linear gradient of acetonitrile from 10% to 60% in trifluoroacetic acid; 50 min Detection: Absorbance at 214 nm (analysis) Column: Beckman Ultrabore RPSC (φ4.6x
75 mm) Flow rate: 1 ml/min Elution: Same conditions as preparation Retention time: 11.9 minutes The retention time on IPLc of α-hANP obtained in this example was that of α-hANP standard product (sold by Funakoshi Co., Ltd.). ) was consistent with that of

(4) Amino acid analysis sample of α-hANP is reduced, carboxymethylated, and then hydrolyzed with 6N hydrochloric acid at 110° C. for 24 hours. The amino acid composition of α-hANP was determined using an amine acid analysis system manufactured by Waters.

α-hANPf7) Amino acid composition (residues per mol) agreed with the calculated values.

(5) Amino acid sequence analysis of α-hANP The N-terminal amino acid sequence of α-hANP was analyzed using the Edman method (DABITC method).
[described in FEBS Letters, Record, 205 (1978)], and the N-terminal Ser and Leu sequences were confirmed. C-terminal amino acid (Ser-Pha-Arg-r
yr) was determined by digestion with carboxypeptidase followed by amino acid analysis using a Waters amino acid analysis system.Then, the entire amino acid sequence of α-hANP obtained in the example was determined using both methods. It was confirmed that α-hANP was obtained.

Construction and cloning of plasmid pBR322trpss (shown in Figure 18) Plasmid pBR322 is digested with EcoRI and C1aI. Large fragment (4340b, p, ) 0.8
% agarose gel electrophoresis and ligated with the synthetic trp promoter ■ gene in the presence of T4 DNA ligase and 1 mM ATP. This ligation mixture is used to transform T. coli HBIOI. Plasmid DNA (pBR322tr
pss) is isolated from the transformed clones and qualitatively analyzed by restriction endonuclease analysis.

Analysis data: Hpa I; 4445bp, C1a
I-Pst I; Construction and cloning of the 34 bp
Digest with amHI. Small fragment (407b, p, )
isolate. On the other hand, pBR322trpss was C1aI
and BamHI.

The large fragment (4093b, p, ) was isolated and the DN
Ligate with A(407b,p,). After transforming E. coli IBIOI with this ligation mixture, the desired plasmid (pcLaHtr
p-2) was isolated from transformed clones and confirmed by restriction endonuclease analysis. C1aI-Ps
tI (834b, p, ), C1aI-BamHI
(407b, p, ).

Example 18 Synthesis of oligocleotide: According to the method of Example 1, the following oligocleotide is prepared.

(1) )lOGpApTpCpCprpCpGpA
pGpApTpCpApAOH (Tl) (2) HO
GpCpCpTpTpTpApApTplpGpApT
pCpIpCpGpApGo)i (T2) (3) HOTpTpApApApGpGpCpTp
CpCpIpIpTpGpGpAOH (T3) (4) HOApApApApApGpGpCpT
pCpCpApApApApGpGpAOl ((T4) (5) HOGpCpCpTpTpTpIpTpT
pTplpTpTpGOH (T5) (6) HOTp
CpGpApCpApApApApAOH (T6) Example 19 Construction and cloning of a synthetic fd phage terminator (shown in Figures 20 and 21): A synthetic fd phage terminator is constructed similarly to the method described in Example 7 ( (shown in Figure 20).

That is, DNA oligomers T2, T3, T4 and T5 (
0.4 nmol of each) and phosphorylated with T4 polynucleotide kinase in the presence of 1 mM ATP.

Heat the reaction mixture at 6510 for 10 minutes to inactivate the enzyme. Add DNA oligomers T1 and T6 (0.8 nmol each) and I4 DNA ligase to the resulting mixture. The mixture was incubated at 15 °C for 30 min and 2-1
subjected to 6% gradient polyacrylamide gel electrophoresis,
The desired DNA fragment (47b, p, ) was recovered by electroelution and pBR3 digested with BamHI and 5al).
22 large fragments (4088b, p, ) and ligate with the insert. This ligation mixture allows E,
After transforming E. coli HBlol, the desired plasmid (pter21) was isolated from the transformed clone. Restriction enzyme analysis: BamHI-5alI (47b
, p, ), AvaI (817b, p, ).

Example 20 α-hANP expression vector plasmid pcLaHtrp
Construction and cloning of 3t (shown in Figure 22) Niblasmid pCLaHtrp-2 was cloned with PstI and 13
Digest with amHI. This digestion mixture contains smaller fragments (
1241b,p,) and pter21 was isolated by PstI
and pter21 (7
) Ligate to the large fragment (3005b, p, ).

In this ligation mixture, E-, coliHBlol
to obtain transformants E. coli H2. Plasmid CLaHtrp3t (containing the CLaH protein gene) obtained from the RAmp transformant (device H2) was confirmed by restriction endonuclease analysis. C1aI-EcoRI (93b, p, 19
8b, p, ), HindllI-BamHI (1
34b,p,) and PstI-C1aI-XhoI
(834b, p, 411b, p,).

Production of α-hANP using H2: Mutual,
α-hANP is obtained in the same manner as in Examples 14 and 15 using E. coli H2 instead of E. coli H1.

As a result of amino acid sequence analysis, it was confirmed that the amino acid sequence of α-hANP thus obtained matched that of known α-hANP.

[Brief explanation of drawings]

Figures 1-3 show synthetic trp promoter I gene, synthetic tr
The DNA sequences of the p promoter ■ gene and the synthetic trp promoter ■ gene are shown, respectively. Figures 4 and 5 show the DNA sequences and the corresponding amino acid sequences of the peptide CLa gene and the α-hANP gene with linker DNA, respectively. Figure 6 shows the construction of the synthetic trp promoter ■ gene.
The figures show molecular cloning of the synthetic trp promoter ■ gene, respectively. Figure 8 shows the trp promoter vector (pBR322t
rp) construction and cloning. Figure 9 shows the construction of a synthetic trp promoter ■ gene. Figure 10 shows the double trp promoter vector (p32
2dtrps) construction and cloning. Figures 11 and 12 show the D of the synthetic trp promoter.
The construction of a peptide Cd gene having a portion of an NA fragment and its DNA sequence are shown. Figure 13 shows the construction and cloning of plasmid pcci7. Figure 14 shows the construction and cloning of plasmid pCd7trpsd. Figure 15 shows the construction of the α-hANP gene with linker DNA. Figure 16 shows a-hANP expression vector pcLaHtrp
sd construction and cloning. Figure 17 shows the DNA sequence of the CLaH protein gene. Figure 18 shows the construction and cloning of plasmid pBR322trpss. Figure 19 shows the construction and cloning of plasmid pCLaHtrp-2. Figures 20 and 21 show the construction and cloning of synthetic fd phage terminators, respectively. Figure 22 shows the construction and cloning of plasmid pCLaHtrp3t. Figure 1 EcoR engineering 3'-ACGGCTGTAG TATTGCCAAGA[
2CGTTATAAGACTT'l'ACTCG-E
coR engineering AAGGGTATCG-3' To TCCCATAGCTT-51 Fig. 2 EcoR engineering 3'-ACGGCTGTAGTATTGCCAAGAC
CGTTTATAAGACTTTACTCG-EcoR
am) I engineering ACTTCGTGTTGATAG-3'TGMGCAC
MCTATCCTAG-5'Figure 17Thr Leu Phe Leu Gly 1e Le
u Lys Asn Trp LysACT CT'r
TTCTTA GGCA'l"r TTG AAG
AAT TGG AAATGA GAA AAG AA
T CCG TAA AACTTCTTA ACCT'
：'T 工la Val Ser Phe Tyr Ph
e Lys Leu Glu Van, GluATT
GTCTCCTTCTACTTCAAG CTT G
AA GT'r GAGTAA CAG AGG AA
G ATG AAG TTCGAA CTT CM C
TCLeu Arg Arg Ser Ser Cys
Phe Gly Gly krg MetCTG C
GT AGA TCCTCT TGCTTT GGT
GGCCGT ATGGACGCA TCT AGG
AGA ACG AAA CCA CCG GCA T
ACAsn Ser Phe Arg TyrMCTC
T TTCCGT TAC-3'TTG AGA AA
G GCA ATG -5'Glu Glu Se
r Asp Arg Lys 工 le Me
t Gln Ser GinGAG GAG A
GT GACAGA AAA ATA ATG CAG
AGCCAAACTCCTCTCA CTG TCT
TTT TAT TACGTCTCG GTTHis
Glu Phe Gly Met Gly Gly G
lu Ala Lys 5erCAT GAA TTC
GGT ATG GGCGGT GAA GCT A
AA TCTGTA CTT AAG CCA TAC
CCG CCA CTT CGA TTT AGAAs
p Arg Engineering 1a Gly Ala Glr, Ser
Gly Leu Gly CysGACCGCATC
GGT GCT CAG 'TCCGGT CTG G
GCTGTCTG GCG TAG CCA CGA
GTCAGG CCA GACCCG ACA city giant ↓ “He

Claims (17)

[Claims] (1) Amino acid sequence of α-hANP: [There is an amino acid sequence] A synthetic gene encoding. (2) DNA sequence: Coding strand: 5'-TCT CTG CGT AGA
TCC non-encoding chain: 3'-AGA GAC GC
A synthetic gene according to claim 1, which is represented by A TCT AGG [gene sequence is present]. (3) Amino acid sequence of α-hANP: [There is an amino acid sequence] A recombinant vector containing a synthetic gene encoding. (4) DNA sequence: Coding strand: 5'-TCT CTG CGT AGA
TCC non-encoding chain: 3'-AGA GAC GC
A recombinant vector according to claim 3, which contains a synthetic gene represented by A TCT AGG [gene sequence is present]. (5) Amino acid sequence of α-hANP: [There is an amino acid sequence] A transformant containing a synthetic gene encoding. (6) DNA sequence: Coding strand: 5'-TCT CTG CGT AGA
TCC non-encoding chain: 3'-AGA GAC GC
A transformant according to claim 5, which contains a synthetic gene represented by A TCT AGG [gene sequence is present]. (7) A synthetic protected peptide gene having a DNA sequence encoding lysine as the C-terminus of the protected peptide. (8) The synthetic protected peptide gene according to claim 7, which is represented by the DNA sequence shown in FIG. (9) A microorganism transformed with an expression vector containing a synthetic gene encoding the amino acid sequence of the protected peptide-fused α-hANP is cultured in a medium, and the protected peptide-fused α-hANP is collected from the resulting culture. A method for producing α-hANP, which comprises removing the protected peptide portion of protected peptide-fused α-hANP. (10) The method for producing α-hANP according to claim 9, wherein the microorganism is a bacterium. (11) The method for producing α-hANP according to claim 10, wherein the bacterium belongs to the genus Escherichia. (12) The method for producing α-hANP according to claim 11, wherein the bacterium belonging to the genus Escherichia is Escherichia coli. (13) The α-hANP gene part of the synthetic gene is DN
A sequence: Coding strand: 5'-TCT CTG CGT AGA
TCC non-encoding chain: 3'-AGA GAC GC
A method for producing α-hANP according to claim 9, which is represented by A TCT AGG [gene sequence is present]. (14) The method for producing α-hANP according to claim 9, wherein the protected peptide-fused α-hANP has the amino acid sequence shown in FIG. (15) The method for producing α-hANP according to claim 9, wherein the protected peptide portion of the protected peptide-fused α-hANP is removed in the presence of API. (16) Method for cleaving a fusion protein consisting of a peptide having lysine between the protected peptide and the target peptide in the presence of API (17) Synthetic trp promoter III shown in the DNA sequence of FIG.