JPH0516835B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to α-human atrial natriuretic polypeptide (α-human atrial natriuretic polypeptide).
polypeptide, hereinafter abbreviated as “α-hANP”)
This invention relates to a novel fusion protein. More specifically, C.
α-hANP fused via the lysine to a protected peptide whose terminal end is lysine, and then α-hANP
The present invention relates to a method for producing hANNP. PRIOR ART AND PROBLEMS TO BE SOLVED BY THIS INVENTION α-hANP is a known polypeptide that has diuretic effects, natriuresis increasing effects, and antihypertensive effects, and is useful as a diuretic antihypertensive agent. (see Biochemical and Biophysical Research Communications Vol. 118, p. 131 (1984)). Therefore, a method for producing α-hANP with good yield has been desired. Structure and Effects of the Invention The inventors have proposed the production of α-hANP by recombinant DNA technology using an expression vector containing a gene encoding α-hANP fused via the lysine to a protected peptide whose C-terminus is lysine. Created a new law. 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 gene encoding the amino acid sequence of α-hANP fused via the lysine to a protected peptide whose C-terminus is lysine is cultured in a medium; (2) Collect the protected peptide-fused α-hANP from the resulting culture, and (3) collect the protected peptide-fused α-hANP.
The protected peptide part of hANP was
α-hANP is produced by removal in the presence of protease (API). Below, the details of the above manufacturing method will be explained in more detail. Microorganisms to be transformed refer to host cells and may include cells, fungi, human and animal cultured cells, and plant cultured cells. Preferred examples of microorganisms include bacteria, especially for example E.
coliHB101 (ATCC33694), E.coli294
(ATCC31446) and E.colix1776 (ATCC31537), which belong to the genus Escherichia. Expression vectors usually contain at least a promoter and
It consists of a DNA having an operator region, a start codon, 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 Shein-Dalgarno (SD) sequence, such as AAGG. SD
The distance between the sequence and the start codon is preferably 8 to 12
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. promoter·
Examples of the operator region include, in addition to synthetic promoter/operator regions, commonly used promoter/operator regions such as the lactose operon, PL-promoter, and trp-promoter. A preferred example of the promoter/operator region is the synthetic trp promoter newly synthesized by the present inventors,
, and, whose DNA sequence is the first
2 and 3. In the production method of this invention, one to three promoter-operator regions may be used for each expression vector. A preferred initiation codon is the methionine codon (ATG). The protective peptide gene has a DNA sequence corresponding to the amino acid sequence of a peptide or protein that can form a fusion protein with α-hANP and suppresses undesirable degradation of the fusion protein in host cells or cultures. Examples include genes. A preferable example is a "peptide Cd gene" linked to an "LH protein gene" (hereinafter referred to as "peptide Cd gene linked to an LH protein gene").
The DNA sequence is shown in Figure 4. The DNA sequence of the α-hANP gene is α-hANP
from the amino acid sequence of , according to a number of non-trivial criteria. An example of a preferred DNA sequence of the α-hANP gene is shown in FIG. In the Examples described later, the amino acid lysine is encoded between the α-hANP gene and the protected peptide gene.
A DNA sequence was inserted. In this way, the junction of the fusion protein can be cleaved with Achromobacter protease. Examples of the termination codon include commonly used termination codons such as TAG and TGA. A replicable unit is one that is capable of replicating the entire DNA sequence it belongs to in a host cell.
It refers to a DNA sequence, and includes natural plasmids, artificially modified plasmids such as DNA fragments prepared from natural plasmids, and synthetic plasmids. 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 a natural or synthetic terminator, such as a synthetic fd phage terminator. Promoter operator region, start codon, protected peptide gene whose C-terminus is lysine,
When synthesizing the α-hANP gene, stop codon, terminator, etc., the synthesis method can be performed by a known method commonly used for producing polynucleotides. Promoter operator gene, start codon, protected peptide gene whose C-terminus is lysine,
The α-hANP gene and stop codon can be isolated using conventional methods (e.g. restriction enzyme digestion, phosphorylation using T4 polynucleotide kinase, etc.) using appropriate DNA fragments (e.g. linkers, other restriction sites, etc.) if desired. An expression vector can be obtained by continuous and circular ligation with an appropriate replicable unit (plasmid) by ligation using T4 DNA ligase, etc.). The expression vector is transformed into a microorganism (host cell) using a conventional method.
can be inserted into. Transformants can be obtained by insertion by conventional methods such as transformation and microinjection. In the production method of the present invention, α-hANP is produced by culturing the transformant containing the expression vector thus obtained in a medium. The medium may be any medium that allows the transformants 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 bactotryptone. , beef extract and, if desired, inorganic salts such as sodium or potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride, e.g. vitamin B ₁ Other ingredients may also be added to the medium, such as vitamins such as, antibiotics such as ampicillin. The transformant is usually cultured at PH5.5-8.5 (preferably PH7-7.5) and 18-40°C (preferably 25-38°C).
°C) for 5 to 50 hours. The thus-produced protected peptide whose C-terminus is lysine and the α-
Since hANP is normally present in cells of cultured transformants, cells are collected by filtration or centrifugation, and their cell walls and/or cell membranes are removed by conventional methods (e.g., treatment with ultrasound and/or lysozyme, etc.). ) to obtain broken cells. From this destroyed material, the conventional methods (e.g. 8M urea aqueous solution,
Dissolving the protein with a suitable solvent such as 6M guanidine,
α-hANP fused to a protected peptide whose C-terminus is lysine via the lysine can be purified and isolated by dialysis, gel filtration, column chromatography, high-performance liquid chromatography, etc.). α-hANP is a protective peptide-fused α-hANP
, Achromobacter protease (API)
It can be prepared by cutting by treatment with. API is a known enzyme [Biochimica et Biofijica Acta, 660, 51 (1981)], but
Fusion protein prepared by recombinant DNA technology
It is not known that API processing can be used to achieve preferable cleavage. This method can be suitably used to cleave a fusion protein consisting of a peptide that has a lysine between the protected peptide and a target peptide that does not have a lysine in its molecule. Cleavage of the fusion protein may be carried out at pH 5-10, 20-40°C (preferably 35-40°C) for 2-15 hours in an aqueous solution such as a buffer solution or urea aqueous solution. In the examples below, the fusion protein is first treated with API in a PH5 buffer containing 8M urea and then in a PH9 buffer containing 4M urea. Under this condition, the fusion protein is cleaved at the lysine site and the produced α-hANP is spontaneously reconstituted. The α-hANP thus produced is purified and isolated from the obtained culture using the conventional method described above. The figures attached to this specification will be explained below. In some of the figures, the oligonucleotides are represented by the symbol --, 〓〓 or 〓〓 (in the symbol, .
(means the 5′-end phosphorylated by T4 polynucleotide kinase), and the blocked oligonucleotides are --, 〓〓, 〓
Indicated by the symbol 〓, 〓〓, 〓〓 or 〓〓 (in the symbol, △ means the ligated position). Unless otherwise specified, in this specification, each of the DNA sequences A, G, C, and T is represented by the formula: [Formula] and [Formula], and the 5'-terminals A, G, C, and T are each of the formula: and and the 3'-terminals A, G, C, and T each have the formula: and means. In the following examples, Ap, Gp, Cp, and Tp each have the formula: and means 3′-terminal AOH, GOH, COH, TOH
are each formula: and means 5′ terminal HOAp, HOGp, HOCp,
HOTp has the following formula: and , and A ^Bz po, G ^iB po, C ^Bz po, Tpo, and TO are each formula: and means. DMTr is dimethoxytrityl and CE is cyanoethyl. Oligonucleotide mono (or di or tri)mers can be prepared using conventional methods such as the method of Hirose et al.
Nucleic Acids and Enzymes 25 , 225 (1980)], and couplings can be prepared, for example, on cellulose or polystyrene polymers by the phosphotriester method [Nuclide Research;
9, 1691 (1981), New York Assisted
Research, 10 , 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 are obvious to those skilled in the art, for example, by referring to the instructions attached to the commercially available enzymes. That's true. Furthermore, in the examples, "polystyrene polymer" means aminomethylated polystyrene HCl, divinylbenzene content 1%, 100-200 mesh (sold by Peptide Institute, Inc.). Example 1
HOCpTpGpCpGpTpApGpApTpCpCpTpCpTO
Synthesis of H(AH7): (1) Synthesis of DMTrOTpoC ^Bz poTO-succinyl polystyrene polymer (i) Preparation of HOTO-succinyl polystyrene polymer: DMTrO-TO-succinyl polystyrene polymer (51.8 mg,
A solution of 5% dichloroacetic acid (DCA) in dichloromethane (2 ml) is added to 10.37 μmol) [prepared by the method described in New York Acids Research 10, 1755 (1982)].
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. The polymer is washed sequentially with dichloromethane (2 ml x 3), methanol (2 ml x 3) and pyridine (2 ml x 3) and dried under a stream of nitrogen to obtain the polymer adduct. (ii) Preparation of DMTrOTpoC ^Bz po−: DMTrOTpoC ^Bz po−CE prepared by the method described in Proteins, Nucleic Acids, and Oxygen 25 , 225 (1980)
(32.4 mg, 8.12 μmol) was added to a triethylamine-acetonitrile mixture (1:
1v/v, 5ml). The phosphodiester dimer thus obtained (DMTrOTpoC ^Bz po-) is dried and water is separated as an azeotrope with pyridine (2 ml x 2). (iii) Coupling: dimer (DMTrOtpoC ^Bz po−) and mesitylenesulfonyl nitrothiazolide (MSTN)
(80 mg) in pyridine (0.5 ml). The solution is added to the reaction syringe along with the polymer adduct and the mixture is shaken at room temperature for 1 hour. The reaction mixture is filtered through a glass filter with a stream of nitrogen and washed with pyridine (2 ml x 3) to give the polymer adduct. (iv) Acetylation of unreacted 5'-hydroxy groups: Pyridine (0.9 ml) and acetic anhydride (0.1 ml) are added to the polymer adduct obtained above and the mixture is shaken for 15 minutes. The reaction solution was filtered through a glass filter, and the resulting polymer was mixed with pyridine (2 ml x 3), methanol (2 ml x 3) and dichloromethane (2 ml x 3).
3) and then dried with a nitrogen stream. This polymer adduct (DMTrOTpoC ^Bz poTO−
Succinyl polystyrene polymer) was used in the next coupling step. (2) Synthesis of DMTrOTpoC ^Bz poC ^Bz potTpoC ^Bz poTO-succinyl polystyrene polymer: Under the same conditions as in (1) above, DMTrOTPoC ^Bz
poTO-succinyl polystyrene polymer
From DMTrOTpoC ^Bz poC ^Bz poCE (44.9mg)
Synthesize DMTrOTpoC ^Bz poC ^Bz poTpoC ^Bz poTO-succinyl polystyrene polymer. (3) DMTrOA ^Bz poG ^iB poA ^Bz poTpoC ^Bz poC ^Bz
Synthesis of poTpoC ^Bz poTO-succinyl polystyrene polymer: Under the same conditions as (1) above, DMTrOTpoC ^Bz
poC ^Bz poTpoC _Bz poTO-succinyl polystyrene polymer and DMTrOA ^Bz poG ^iB poA ^Bz poCE
(48.5 mg), DMTrOA ^Bz poG ^iB poA ^Bz
poTpoC ^Bz poC ^Bz poTpoC ^Bz poTO-succinyl polystyrene polymer is synthesized. (4) DMTrOC ^Bz poG ^iB poTpoA ^Bz poG ^iB poA ^Bz
synthesis of poTpoC ^Bz poC ^Bz poTpoC ^Bz poTO-succinyl polystyrene polymer: DMTrOA ^Bz poG ^iB under the same conditions as in (1) above.
poA ^Bz poTpoC ^Bz poC ^Bz poTpoC ^Bz poTO-succinyl polystyrene polymer and DMTrOC ^Bz poG ^iB
From poTpoCE (45.1mg), DMTrOC ^Bz poG ^iB
poTpoA ^Bz poG ^Bz poTpoC ^Bz poC ^Bz poTpoC ^Bz
Synthesize poTO-succinyl polystyrene polymer. (5) DMTrOC ^Bz poTpoG ^iB poC ^Bz poG ^iB poTpoA ^Bz
poG ^iB poA ^Bz poTpoC ^Bz poC ^Bz poTpoC ^Bz poTO−
Synthesis of succinyl polymer: Under the same conditions as (1) above, DMTrOC ^Bz poG ^iB
poTpoA ^Bz poG ^iB poA ^Bz poTpoC ^Bz poTpoC ^Bz
poTO-succinyl polystyrene polymer and
From DMTrOC ^Bz poTpoG ^iB poCE (45.1mg),
DMTrOC ^Bz poTpoG ^iB poC ^Bz poG ^iB poTpoA ^Bz
poG ^iB poA ^Bz poTpoC ^Bz poC ^Bz poTpoC ^Bz poTO−
Synthesize succinyl polystyrene polymer (60 mg). In this final step, the unreacted 5′-
Hydroxy groups do not need to be protected with acetyl groups. (6)
HOCpTpGpCpGpTpApGpApTpCpCpTpCpT
Synthesis of OH: DMTrOC ^Bz poTpoG ^iB poC ^Bz poG ^iB poTpoA ^Bz
poG ^iB poA ^Bz poTpoC ^Bz poC ^Bz poTpoC ^Bz poTO−
Succinyl polystyrene polymer (60mg),
1M, N,N,N',N'-tetramethyleneguanidium pyridine-2-aldoximate [dioxane-water (1:1 v/v,
1 ml) for 20 hours. 28 to the reaction mixture
% (w/w) aqueous ammonia (12 ml) is added and the mixture is heated at 60° C. for 5 hours. The solid polymer is filtered off and washed with water (10 ml). The filtrate and washings were evaporated to dryness, and the residue was diluted with 80% aqueous acetic acid (25%
ml) for 15 minutes. After removing the solvent, the residue is dissolved in 0.1 M triethylammonium carbonate buffer (PH 7.5, 25 ml) and washed with diethyl ether (3 x 25 ml). Evaporate the aqueous layer to dryness,
When the residue was dissolved in 0.1M triethylammonium carbonate buffer (PH7.5, 2ml), the crude
HOCpTpGpCpGpTpApGpApTpCpCpTpCpT
OH is obtained. (7)
HOCpTpGpCpGpTpApGpApTpCpCpTpCpT
Purification of OH (i) Initial purification of the crude product is performed by Biogel P2 (BioRad) (24 x 2.6 cm ID) column chromatography. The fraction corresponding to the first elution peak (50mM containing 0.1mM MEDTA)
M ammonium acetate, flow rate: 1 ml/min) is collected and lyophilized to obtain the first purified product. (ii) The second purification of the first purification is CDR-10
(Mitsubishi Kasei) (25 cm x 4.6 mm ID) by high performance liquid chromatography (HPLC)
Ammonium acetate - 10% (v/v) from aqueous ethanol to 4.5M ammonium acetate - 10%
(v/v) using a linear gradient to aqueous ethanol (80 min, flow rate: 1 ml/min, 60°C) to obtain a second purified product. (iii) The third purification of the second purified product is reversed phase.
HPLC [Rp-18-5μ (x77) (Merck), 15cm
x 4 mm ID] from 0.1 M ammonium acetate to 0.1 M ammonium acetate - 15% (v/v)
A linear gradient to aqueous acetonitrile (40 min, 1.5 ml/min, room temperature) is used to obtain the final purified product. (
HOCpTPGpCpGpTpApGpApTpCpCPTpCpTO
H (8) Oligonucleotide (
HOCpTpGpCpGpTpApGpApTpCpCpTpCpT
(i) Digestion with phosphodiesterase
HOCpTpGpCpGpTpApGpApTpCpCpTpC
pTOH (10μg, 5.1μ), 0.2M _MgCl2 (20μ
), 0.2M Tris-HCl (PH8.5) (20μ) and 0.1mM EDTA (144.9μ) with phosphodiesterase (10 units, 10μ).
℃ for 20 minutes and then heated to 100℃ for 2 hours. (ii) HPLC analysis The oligonucleotides in the reaction mixture are
HPLC [CDR-10 (Mitsubishi Kasei), 25cm x 4.6mm
ID] from water to 2.0M ammonium acetate (PH
3.4) linear gradient (40 minutes, flow rate: 1.5ml/
min, 60°C). 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 values: pCHO3.770, pAOH2.026,
pTOH5.237, pGOH2.968 Example 2 Synthesis of oligonucleotides: The following oligonucleotides are prepared in the same manner as described in Example 1. [Table] Example 3 Synthesis of oligonucleotides: The following oligonucleotides are prepared in the same manner as described in Example 1. [Table] [Table] Example 4 Synthesis of oligonucleotides: The following oligonucleotides are prepared in the same manner as described in Example 1. [Table] Example 5 Construction and cloning of a synthetic trp promoter gene (as shown in Figures 6 and 7): The trp promoter gene is constructed in a manner similar to that described in Example 7 (as shown in Figure 6).
The synthetic gene was ligated with the EcoRI-BamHI fragment of pBR322 (commercially available from Takara Shuzo, NEB, etc.), followed by E. coli HB101.
(ATCC33694) with the ligation product. A plasmid obtained from a transformant of ^R Amp and ^S Tet was digested with HpaI, and a band (4.1 kpP) was detected by polyacrylamide gel electrophoresis (PAGE).
, followed by digestion with BamHI and PAGE
A band of 90 b.p. was confirmed. Furthermore, EcoRI−
The 56 b.p. fragment resulting from BamHI digestion was confirmed by PAGE and comparison with size markers. This plasmid was named pTrpEB7 and used to construct an expression vector. Example 6 Construction and cloning of trp promoter vector (pBR322trp) (shown in Figure 8): Plasmid pBR322 (9 μg) was incubated with EcoRI and
Digest with BamHI restriction endonuclease. 65
The reaction was stopped by heating at °C for 5 min, and the fragments were separated by 0.8% agarose gel electrophoresis and 375b.
Obtain a small fragment (500 ng) of p. Meanwhile, plasmid pTrpEB7 (10 μg) was digested with EcoRI and BamHI, followed by preparative gel electrophoresis, and 4094b.
Obtain a large fragment (5 μg) of p. pTrpEB7
EcoRI-BamHI fragment (4094b.p., 200μg)
EcoRI-BamHI fragment of pBR322 (375b.p., 100n
g) and ligation buffer [50mM Tris
-HCl (PH7.6), 10mM _MgCl2 , 20mM DTT,
1mM ATP, 1mM spermidine, 50μg/ml
BSA] (20μ) at 15°C overnight. E.coliHB101 in this ligation mixture
were transformed by Kushiner's method [T. Maniatis et al., Molecular Cloning, p252 (1982), Cold Spring Harbor Laboratory] and transformed to be resistant to tetracycline on plates containing tetracycline (25 μg/ml). Get a body. Plasmid pBR322trp isolated from the transformant was incubated with EcoRI-BamHI (375b.p.,
4094b.p.) and HpaI (4469b.p.), 7.5
The trp promoter gene was confirmed by %PAGE and 0.8% agarose gel electrophoresis. Example 7 Construction of a synthetic trp promoter gene (shown in Figure 9): Block', ' and ' oligonucleotides (B-M') (2.0 nmol each) were injected into T4 polynucleotide kinase (BRL, 2.5 units). ) in a ligation buffer (70 μ) at 37°C.
Time phosphorylation. Add T4 DNA ligase (300 units) and 20 m
MATP (2 μ) is added and the mixture is incubated at 15 °C for 30 min, then heated at 65 °C for 10 min to stop the reaction. The reaction mixtures of each of these blocks (′,′ and′) are combined and mixed with non-phosphorylated oligonucleotides (A,N′) in the presence of T4 DNA ligase (360 units) and 20mM ATP (2μ). . After incubating the mixture for 1 hour at 15°C, the final ligation product was purified by 2-16% gradient polyacrylamide gel electrophoresis (PAGE) to yield 106 b.p.
Obtain a synthetic trp promoter gene. Example 8 Double trp promoter vector (p322dtrpS)
Construction and cloning (shown in Figure 10): Plasmid pBR322trp was digested with EcoRI and ClaI, followed by preparative agarose gel electrophoresis.
Obtain a large fragment of 4446b.p. This fragment (4446b.
p.) is ligated with the trp promoter gene (106 b.p.) obtained in Example 7 in the presence of T4 DNA.
E. coli HB101 is transformed with this ligation mixture to obtain transformants resistant to ampicillin and tetracycline. Plasmid p322dtrpS obtained from this transformant was confirmed by restriction endonuclease analysis. ClaI−BamHI
(325b.p.), HpaI (107b.p.) and Aat−ClaI
(287b.p.). Example 9 Construction of a peptide Cd gene with a portion of the DNA fragment of a synthetic trp promoter (shown in Figures 11 and 12): Block ``,'' and '' oligonucleotides (0.2 nmol) (Np1-Cd8 , shown in Example 4) using T4 polynucleotide kinase (2.5 units) and ligation buffer (60μ
) for 1 hour at 37°C. Add T4 DNA ligase (360 units) and 20mMATP (2μ) to the reaction mixture of each block, and mix the mixture.
Incubate for 1 hour at 15°C. The reaction mixtures of each block were combined and combined with T4 DNA ligase (360 units) and 20 mMATP (2 μ) for 15 min.
Incubate overnight at 80 °C, followed by 10 min at 80 °C.
Heat for a minute. Add 500mM aqueous sodium chloride solution (20μ) and EcoRI (20 units) to the mixture. After incubation for 2 hours at 37°C, 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 the 12th
As shown in the figure. Example 10 Construction and cloning of plasmid pCdγ (shown in Figure 13): Plasmid pγtrp (4544b.p.) (E.coli F-9 containing this plasmid has been deposited with FIKEN as FERM BP-905). (see UK Patent Application Publication No. 2164650) was digested with HpaI and EcoRI to obtain a large fragment (4510 b.p.). This is ligated to the peptide Cd gene containing a portion of the synthetic trp promoter DNA fragment obtained in Example 9 in the presence of T4 DNA ligase. This ligation mixture is used to transform E. coli HB101. ^R
The plasmid (pCdγ) obtained from the Amp transformant was confirmed by restriction endonuclease analysis. ClaI−BamHI (543b.p.), ClaI−Hind
(273b.p.), ClaI−EcoRI (93b.p.) and Aat
−ClaI (180b.p.). Example 11 Construction and cloning of plasmid pCdγtrpSd (shown in Figure 14): Plasmid pCdγ was digested with ClaI and BamHI to obtain a small fragment (543 b.p.), which was transformed into p322dtrpSd.
(Example 7) ClaI-BamHI fragment (4223 b.p.) and
Ligate in the presence of T4 DNA ligase. Transform E. coli HB101 with this ligation mixture. The plasmid (pCdγtrpSd) obtained from ^{the R} Amp transformant was confirmed by restriction endonuclease analysis. HpaI−BamHI (1075, 575b.
p.), ClaI−BamHI (543b.p.), PstI−EcoRI
(1057b.p.), EcoRI−BamHI (450b.p.), Hind
−BamHI (270b.p.) and ClaI−Hiand)
273 b.p.). Example 12 Preparation of α-hANP gene with linker DNA (shown in Figure 15): Block and oligonucleotides (AH2-AH17) (0.2 mol each) were prepared using T4 polynucleotide kinase (2.5 units). Phosphorylate for 1 hour at 37°C in ligation buffer (70μ). Add T4 DNA ligase (300 units) and 20mM to the reaction mixture for each block.
ATP (2μ) is added and the mixture is incubated at 15°C for 30 minutes. Stop the reaction by heating at 65 °C for 10 min. The reaction mixtures of the two blocks (and) were combined and the non-phosphorylated oligonucleotides (AH1,
Mix with AH18). After incubating the mixture for 1 hour at 15°C, the final ligation product was purified by 2-16% gradient PAGE to yield a 134 b.p. α-hANP gene with linker DNA (see Figure 5). show). Example 13 Construction and cloning of α-hANP expression vector pCLaHtrpSd (shown in Figure 16): Plasmid pCdγtrSd was transformed into Hind and BamHI
A large fragment (4743b.p.) was obtained by digestion with α-hANP gene (134b.p.) with linker DNA.
and ligate in the presence of T4 DNA ligase.
E.coliHB101 using this ligation mixture
to obtain transformants E and coliH1. ^R
A plasmid (pCLaHtrpSd) obtained from a transformant of Amp (E.coliH1) [CLa protein (peptide
It contained the CLa fusion α-hANP protein gene, the DNA sequence of which is shown in FIG. 17] was confirmed by restriction endonuclease analysis. Aat
−ClaI (287b.p.), ClaI−BamHI (407b.p.), ClaI
−EcoRI (93, 198b.p.), EcoRI−BamHI (116,
198b.p.), Hind−BamHI (134b.p.) and
HpaI−BamHI (107, 439 b.p.). Example 14 Expression of gene encoding peptide CLa fusion α-hNAP (CLaH protein): L broth (20 ml) containing 50 μg/ml ampicillin
The expression vector (plasmid) was grown overnight in
A culture of E. coliH1 containing pCLaHtrpSd) was
% glucose, 0.5% casamino acids (casein hydrolyzate), 50 μg/ml vitamin B ₁ and 25 μg/ml
Diluted with M-9 medium (400 ml) containing ampicillin,
Culture this E. coli at 37°C. Culture medium A600
When the absorbance (absorbance at 600 nm) is 0.5, β-indoleacrylic acid (2 mg/ml ethanol, 2 ml) is added and the cells are incubated for 3 hours (final A
= 600 = 1.85). Next, the bacterial cells are centrifuged (6000 rpm,
4° C. for 5 minutes). Example 15 Isolation and purification of α-hANP: (1) Peptide CLa-fused α-hANP (CLaH protein)
Separation and purification: The wet bacterial cell paste obtained from the culture solution (600 ml) prepared in Example 14 was diluted with 10 mM PBS-
EDTA (PH7.4) [NaCl (8.0g) in 1 liter,
KCl (0.2g), _Na2HPO4・_{12H2O (} 2.9g),
KH ₂ PO ₄ (0.2 g), EDTA (3.73 g)], and the cells are disrupted by sonication at 0°C.
Centrifuge at 15,000 rpm for 20 minutes (4°C) to collect the pellet, which is dissolved in 6M guanidine-HCl, 10
Suspend in 8 ml of a mixture of mM PBS-EDTA and 2 mM β-mercaptoethanol and sonicate the suspension at 0°C. This suspension
Centrifugation is performed at 15000 rpm for 20 minutes (4°C), and the supernatant is dialyzed against a 10 mM PBS-EDTA solution containing p-nitrophenylmethylsulfonyl fluoride (PMSF) at 4°C overnight. After centrifugation of the dialyzed fraction (15,000 rpm, 4°C, 20 min), the pellet was dissolved in 100mM guanidine-HCl, 10mM EDTA, and 10mM dithiothreitol.
Dissolved in Tris-HCl buffer (PH8.0) (8 ml),
Leave the solution overnight. This solution was dialyzed twice against 1M acetic acid (0.5 liters) containing 10mM 2-mercaptoethanol, and then dialyzed twice with trisaminomethane.
Adjust to PH8.0. The resulting precipitate (fusion protein;
15.2mg) was collected by centrifugation (3000 rpm, 10 minutes) and added to 10mM sodium acetate buffer (PH5.0).
Wash with (2) Achromobacter protease (AP)
Desorption of peptide CLa from peptide CLa-fused α-hANP by:
Suspend in mM sodium acetate buffer (PH5.0) (30 ml) and incubate the suspension with Achromobacter protease (AP) (0.25 units) (Wako Pure Chemical Industries, Ltd.) at 37° C. for 2 hours. The reaction mixture was diluted with distilled water (30 ml), adjusted to PH9.0 with trisaminomethane, then diluted with AP (0.25
Add additional amount (unit) and incubate at 37°C for 2 hours. The reaction solution was diluted with 10mM sodium phosphate buffer (PH7.0) (120ml) and adjusted to pH7 with acetic acid.
Adjust to. This solution is applied to a Sp-Sephadex C-25 column (15 ml) equilibrated with 10 mM sodium phosphate buffer (PH 7.0). The column was washed with the same buffer and eluted with 10mM sodium phosphate buffer (PH8.0) containing 0.5M aqueous sodium chloride to obtain partially purified α-
Collect fractions containing hANP (0.4 mg). (3) High performance liquid chromatography (HPLC): The pooled fractions obtained in the same manner as in (2) above were concentrated under reduced pressure, dialyzed against water (300 ml),
Purified by reverse-phase HPLC to obtain pure α-hANP (0.3
mg). HPLC conditions (preparation) Column: Semi-preparative Beckman Ultrapore (φ10 x 250 mm) Flow rate: 2.5 ml/min Elution: 10% to 60% in 0.01M trifluoroacetic acid
Linear gradient of acetonitrile up to 50 minutes Detection: Absorbance at 214 nm (analysis) Column: Becman Ultrapore (RPSC
(φ1.6×75 mm) Flow rate: 1 ml/min Elution: Same conditions as preparation Retention time: 11.9 min
The retention time on HPLC was consistent with that of α-hANP standard product (sold by Funakoshi). (4) Amino acid analysis of α-hANP The sample was reduced, carboxymethylated, and then
Hydrolyze with 6N hydrochloric acid at 110℃ for 24 hours. α
-The amino acid composition of hANP was determined using an amino acid analysis system manufactured by Waters. The amino acid composition (residues per mol) of α-hANP agreed with the calculated value. (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) [FEBS Letters, 93 , 205
(1978)], and the N-terminal Ser
and Leu sequences were confirmed. The C-terminal amino acid (Ser-Phe-Arg-Tyr) was determined by digestion with carboxypeptidase followed by amino acid analysis using a Waters amino acid analysis system. Next, the entire amino acid sequence of α-hANP obtained in the example was determined using both methods, and it was confirmed that α-hANP was obtained. Example 16 Construction and cloning of plasmid pBR322trpSs (shown in Figure 18): Plasmid pBR322 is digested with EcoRI and ClaI. The large fragment (4340 b.p.) is purified by 0.8% agarose gel electrophoresis and ligated with the synthetic trp promoter gene in the presence of T4 DNA ligase and 1mM ATP. This ligation mixture is used to transform E. coli HB101.
Plasmid DNA (pBR322trpSs) is isolated from the transformed clone and qualitatively analyzed by restriction endonuclease analysis. Analysis data: Hpa; 4445bp, ClaI−Pst
;834bp Example 17 Construction and cloning of plasmid pCLaHtrp-2 (shown in Figure 19): Plasmid pCLaHtrpSd was incubated with ClaI and BamHI.
Digest it with. Isolate a small fragment (407 b.p.).
Meanwhile, pBR322trpSs is digested with ClaI and BamHI. Separate the large fragment (4093 b.p.) and
Ligate with DNA (407b.p.). After transforming E. coli HB101 with this ligation mixture, the desired plasmid (pCLaHtrp-2) was isolated from the transformed clone and confirmed by restriction endonuclease analysis. ClaI−PstI
(834b.p.), ClaI−BamHI (407b.p.). Example 18 Synthesis of oligonucleotide: According to the method of Example 1, the following oligonucleotide is prepared. (1)
HOGpApTpCpCpTpCpGpApGpApTpCpApAO
H (T1) (2)
HOGpCpCpTpTpTpApApTpTpGpAptpCpTp
CpGpApGOH (T2) (3)
HOTPTpApApApGpGpCpTpCpCpTpTpTpTp
GpGpAOH (T3) (4)
HOApApApApApGpGpCpTpCpCpApApApAp
GpGpAOH (T4) (5)
HOGpCpCpTpTpTpTpTpTpTpTpTpTpGOH
(T5) (6) HOTpCpGpApCpApApApApAOH (T6) Example 19 Construction and cloning of synthetic fd phage terminators (shown in Figures 20 and 21): Synthetic fd phage terminators were synthesized similarly to the method described in Example 7. (shown in Figure 20). i.e. DNA oligomers T2, T3, T4 and T5
(0.4 nmol each) and phosphorylated with T4 polynucleotide kinase in the presence of 1 mM ATP. Heat the reaction mixture at 65 °C for 10 min to inactivate the enzyme. Add DNA oligomers T1 and T6 (0.8 nmol each) and T4 DNA ligase to the resulting mixture. The mixture was incubated at 15°C for 30 min, subjected to 2-16% gradient polyacrylamide gel electrophoresis, and the desired DAN fragment (47 b.p.) was recovered by electroelution and pBR322 digested with BamHI and SalI. Insert and ligate into a large fragment (4088b.p.). After transforming E. coli HB101 with this ligation mixture, the desired plasmid (pter21) was isolated from the transformed clone. Restriction enzyme analysis: BamHI−SalI (47b.p.),
AvaI (817b.p.). Example 20 α-hANP expression vector plasmid
Construction and cloning of pCLaHtrp3t (22nd
(shown in the figure): Plasmid pCLaHtrp-2 was incubated with PstI and
Digest with BamHI. A smaller fragment (1241 b.p.) was isolated from this digestion mixture, and pter21 was digested with PstI and
Ligate to the large fragment of pter21 (3005 b.p.) obtained by digestion with BamHI. E.coliHB101 is transformed with this ligation mixture to obtain a transformant E.coliH2. Plasmid CLaHtrp3t (containing the CLaH protein gene) obtained from the ^R Amp transformant (E. coli H2) was confirmed by restriction endonuclease analysis.
ClaI−EcoRI (93b.p., 198b.p.), Hind−
BamHI (134b.p.) and PstI−ClaI−Xho
(834b.p., 411b.p.). Example 21 Production of α-hANP using E.coli H2: Example using E.coli H2 instead of E.coli H1
Obtain α-hANP in the same manner as 14 and 15. 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 the drawing]

Figures 1 to 3 show the synthetic trp promoter gene,
The DNA sequences of the synthetic trp promoter gene and the synthetic trp promoter gene are shown, respectively. Fourth
The figure and FIG. 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. FIG. 6 shows the construction of a synthetic trp promoter gene, and FIG. 7 shows the molecular cloning of the synthetic trp promoter gene. Figure 8 shows the trp promoter vector (pBR322trp), construction and cloning. Figure 9 shows the construction of a synthetic trp promoter gene. Figure 10 shows the construction and cloning of the double trp promoter vector (p322dtrpS). Figures 11 and 12 are composite trp
Peptide with part of promoter DAN fragment
The construction of the Cd gene and its DNA sequence are shown, respectively. Figure 13 shows the construction and cloning of plasmid pCdγ. Figure 14 is a plasmid
Construction and cloning of pCdγtrpSd is shown. Figure 15 shows the construction of the α-hANP gene with linker DNA. Figure 16 shows the construction and cloning of the α-hANP expression vector pCLaHtrpSd. Number 17 shows the DNA sequence of the CLaH protein gene. Figure 18 shows the construction and cloning of plasmid pBR322trpSs. Figure 19 is a plasmid
Construction and cloning of pCLaHtrp-2 is shown. 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.

Claims (1)

[Scope of Claims] 1. A method for producing α-hANP, which comprises cleaving α-hANP fused via the lysine to a protected peptide whose C-terminus is lysine in the presence of Achromobacter protease. 2 A microorganism transformed with an expression vector containing a gene encoding α-hANP fused via the lysine to a protective peptide whose C-terminus is lysine is cultured in a medium, and the fusion protein is extracted from the resulting culture. The method for producing α-hANP according to claim 1, characterized in that the protected peptide is removed in the presence of Achromobacter protease.