Process for the production of alpha-human atrial natriuretic polypeptide

Abstract

The present invention relates to a process for the production of .alpha.-human atrial natriuretic polypeptide by recombinant DNA technology.

Description

[0001] This invention relates to a new process for the production of .alpha.-human atrial natriuretic polypeptide (hereinafter referred to as the abbreviation ".alpha.-hANP") by recombinant DNA technology. More particularly, it relates to a new process for the production of .alpha.-hANP by recombinant DNA technology, to chemically synthesized genes for .alpha.-hANP and protective peptide-fused .alpha.-hANP and to a corresponding recombinant vector and transformant comprising the same.

[0002] The .alpha.-hANP is a known polypeptide having a diuretic, natriuretic, vasorelaxant and antihypertensive activities. Therefore, it may be useful in clinical treatment of hypertension as antihypertensive diuretic agent and has the following structure: 1

[0003] (Cf. Biochemical and Biophysical Research Communications Vol.118, page 131 (1984)).

[0004] The inventors of this invention have newly created a process for the production of .alpha.-hANP by recombinant DNA technique using an expression vector comprising a synthetic gene encoding the amino acid sequence (I) of .alpha.-hANP. According to this process, .alpha.-hANP can be obtained in high yield.

[0005] This invention provide a process for the production of .alpha.-hANP by (1) culturing a microorganism transformed with an expression vector comprising a synthetic gene encoding an amino acid sequence of a protective peptide-fused .alpha.-hANP in a nutrient medium, (2) recovering the protective peptide-fused .alpha.-hANP from the cultured broth and (3) removing the protective peptide part of the protective peptide-fused .alpha.-hANP.

[0006] In the above process, particulars of which are explained in more detail as follows.

[0007] The microorganism is a host cell and may include bacteria, fungi, cultured human and animal cells and cultured plant cells. Preferred examples of the microorganism may include bacteria especially a strain belonging to the genus Escherichia (e.g. E. coli HB101 (ATCC 33694), E. coli 294 (ATCC 31446), E. coli .chi. 1776 (ATCC 31537), etc).

[0008] The expression vector is usually composed of DNA having at least a promoter-operater region, initiation codon, synthetic protective peptide gene, synthetic .alpha.-hANP gene, termination codon(s) and replicatable unit.

[0009] The promoter-operater region comprises promoter, operater and Shine-Dalgarno (SD) sequence (e.g. AAGG, etc.). The distance between SD sequence and intiation codon is preferably 8-12 b. p. and in the most preferable case as shown in the working Examples mentioned below, the distance between SD sequence and initiation codon (ATG) is 11 b.p. Examples of the promoter-operater region may include conventionally employed promoter-operater region (e.g. lactose-operon, PL-promoter, trp-promoter, etc.) as well as synthetic promoter-operater region. Preferred examples of the promoter-operater region are synthetic trp promoter I, II and III which were newly synthesized by the inventors of this invention and DNA sequences thereof are shown in FIGS. 1, 2 and 3, respectively. In the process, there may be used 1-3 consecutive promoter-operater region(s) per expression vector.

[0010] Preferred initiation codon may include methionine codon (ATG).

[0011] The protective peptide gene may include DNA sequence corresponding to any of peptide or protein which is capable of forming a fused protein with .alpha.-hANP and inhibiting undesired degradation of the fused protein in the host cell or the cultured broth. One of preferred examples is "peptide Cd gene" linked to "LH protein gene" (hereinafter "the peptide Cd gene linked to LH protein gene" is referred to as "peptide CLa gene"), DNA sequence of which is shown in FIG. 4.

[0012] The DNA sequence of .alpha.-hANP gene is designed from the amino acid sequence of .alpha.-hANP, subjected to a number of specific non-obvious criteria. Preferred example of DNA sequence of .alpha.-hANP gene is shown in FIG. 5. In the working Examples as mentioned below, between the .alpha.-hANP gene and the protective peptide gene, a DNA sequence encoding amino acid lysine is inserted, with the purpose of Achromobacter protease I digestion at the junction of the fused protein.

[0013] The termination codon(s) may include conventionally employed termination codon (e.g. TAG, TGA, etc.).

[0014] The replicatable unit is a DNA sequence capable of replicating the whole DNA sequence belonging thereto in the host cells and may include natural plasmid, artificially modified plasmid (e.g. DNA fragment prepared from natural plasmid) and synthetic plasmid and preferred examples of the plasmid may include plasmid pBR 322 or artificially modified thereof (DNA fragment obtained from a suitable restriction enzyme treatment of pBR 322). The replicatable unit may contain natural or synthetic terminator (e.g. synthetic fd phage terminator, etc.).

[0015] Synthetic preparation of promoter-operater region, initiation codon, protective peptide gene, .alpha.-hANP gene and termination codon can be prepared in a conventional manner as generally employed for the preparation of polynucleotides.

[0016] The promoter-operater region, initiation codon, protective peptide gene, .alpha.-hANP gene and termination codon(s) can consecutively and circularly be linked with an adequate replicatable unit (plasmid) together, if desired using an adequate DNA fragment(s) (e.g. linker, other restriction site, etc.) in a conventional manner (e.g. digestion with restriction enzyme, phosphorylation using T4 polynucleotide kinase, ligation using T4 DNA-ligase) to give an expression vector.

[0017] The expression vector can be inserted into a microorganism (host cell). The insertion can be carried out in a conventional manner (e.g. transformation, microinjection, etc.) to give a transformant.

[0018] For the production of .alpha.-hANP in the process of this invention, thus obtained transformant comprising the expression vector is cultured in a nutrient medium.

[0019] The nutrient medium contains carbon source(s) (e.g. glucose, glycerine, mannitol, fructose, lactose, etc.) and inorganic or organic nitrogen source(s) (ammonium sulfate, ammonium chloride, hydrolysate of casein, yeast extract, polypeptone, bactotrypton, beef extracts, etc.). If desired, other nutritious sources (e.g. inorganic salts (e.g. sodium or potassium biphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (e.g. vitamin B1), antibiotics (e.g. ampicillin), etc.) may be added to the medium.

[0020] The culture of transformant may generally be carried out at pH 5.5-8.5 (preferably pH 7-7.5) and 18-40.degree. C. (preferably 25-38.degree. C.) for 5-50 hours.

[0021] Since thus produced protective peptide-fused .alpha.-hANP generally exists in cells of the cultured transformant, the cells are collected by filtration or centrifuge, and cell wall and/or cell membrane thereof is destroyed in a conventional manner (e.g. treatment with super sonic waves and/or lysozyme, etc.) to give debris. From the debris, the protective peptide-fused .alpha.-hANP can be purified and isolated in a conventional manner as generally employed for the purification and isolation of natural or synthetic proteins (e.g. dissolution of protein with an appropriate solvent (e.g. 8M aqueous urea, 6M guanidine, etc.), dialysis, gel filtration, column chromatography, high performance liquid chromatography, etc.).

[0022] The .alpha.-hANP can be prepared by cleaving the protective peptide-fused .alpha.-hANP in the presence of an appropriate protease (e.g. Achromobacter Protease I(AP I), etc.) treatment or chemical method (e.g. treatment with cyanogen bromide). In the case where C-terminal of the protective peptide is lysine, there can preferably be employed treatment with API. Although API is a known enzyme (Cf. Biochim. Biophys. Acta., 660, 51 (1981)), it has never been reported that fused proteins prepared via recombinant DNA technology can preferably be cleaved by the treatment with API. This method may preferably be employed for cleaving a fused protein composed of peptides having a lysine between a protective peptide and a target peptide having no lysine in its molecule.

[0023] The cleavage of the fused protein may be carried out at pH 5-10 and 20-40.degree. C. (preferably 35-40.degree. C.) for 2-15 hours in an aqueous solution (e.g. buffer solution, aqueous urea, etc.).

[0024] In the working Examples as mentioned below, the fused protien is treated with API firstly in a buffer solution containing 8M urea at pH 5, secondly, in a buffer solution containing 4M urea at pH 9. In this condition, the fused protein is cleaved at lysine site, and the produced .alpha.-hANP is refolded spontaneously.

[0025] Thus produced .alpha.-hANP can be purified and isolated from the resultant reaction mixture in a conventional manner as mentioned above.

[0026] The Figures attached to this specification are explained as follows.

[0027] In the some of Figures, oligonucleotides are illustrated with the symbol z,1 or (in this symbol, the mark .circle-solid. means 5'-phosphorylated end by T4 polynucleotide kinase), and blocked oligonucleotides are illustrated with the symbol, or (in this symbol, the mark .DELTA. means ligated position).

[0028] In the DNA sequence in this specification, A, G, C and T mean the formula: 2

[0029] respectively, and

[0030] 5'-terminal A, G, C and T mean the formula: 3

[0031] respectively, and

[0032] 3-terminal A, G, C and T mean the formula; 4

[0033] respectively, unless otherwise indicated.

[0034] In the following Examples, following abbreviations are used.

[0035] Ap, Gp, Cp and Tp mean the formula: 5

[0036] respectively, and

[0037] 3'-teminal AOH, GOH, COH and TOH mean the formula: 6

[0038] respectively, and

[0039] 5'-terminal HOAp, HOGp, HOCp and HOTp mean the formula: 7

[0040] respectively, and

[0041] A.sup.Bzpo, G.sup.iBpo, C.sup.Bzpo, Tpo and TO mean the formula: 89

[0042] respectively, and

[0043] DMTR is dimethoxytrityl, and

[0044] CE is cyanoethyl.

[0045] Mono (or di, or tri)mer (of oligonucleotides) can be prepared by, for examples the Hirose's method [Cf. Tanpakushitsu Kakusan Kohso 25, 255 (1980)] and coupling can be carried out, for examples on cellulose or polystyrene polymer by a phosphotriester method [Cf. Nucleic Acid Research, 9, 1691 (1981), Nucleic Acid Research 10, 1755 (1982)].

[0046] The following Examples are given for the purpose of illustrating this invention, but not limited thereto.

[0047] In the Examples, all of the used enzymes (e.g. restriction enzyme, T4 polynucleotide kinase, T4 DNA ligase) are commercially available and conditions of usage of the enzymes are obvious to the person skilled in the art, for examples, referring to a prescription attached to commercially sold enzymes.

[0048] Further, in the Examples, the term "polystyrene polymer" means aminomethylated polystyrene.HCl, divinylbenzene 1%, 100-200 mesh (sold by Peptide Institute Inc.)

EXAMPLE 1

[0049] Synthesis of HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH (AH7)

[0050] (1) Synthesis of DMTrOTpoC.sup.BzpoTO-succinyl-polystyrene polymer

[0051] i) Preparation of HOTO-succinyl polystyrene polymer:

[0052] To a DMTrO-TO-succinyl-polystyrene polymer (51.8 mg, 10.37.mu. mole) (prepared by the method described in Nucleic Acid Research 10, 1755 (1982)) in a reaction syringe, 5% dichloroacetic acid (DCA) solution in dichloromethane (2 ml) was added. After the standing for 1 minute, the mixture was filtered through filter glass by nitrogen gas. The DCA treatment was repeated more two times. The polymer was washed with dichloromethane (2 ml.times.3), methanol (2 ml.times.3) and pyridine (2 ml.times.3) succesively, and dried by nitrogen gas stream to give polymer adduct I.

[0053] ii) Preparation of DMTrOTpoC.sup.Bzpo-:

[0054] DMTrOTpoC.sup.Bzpo-CE (32.4 mg, 8.12 .mu.mole) prepared by the method described in Tanpakushitsu Kakusan Kohso 25, 255 (1980) was treated with a mixture of triethylamine and acetonitrile (1:1 v/v, 5 ml) at room temperature for 30 minutes. The phosphodiester dimer (DMTrOTpOC.sup.Bzpo-) thus obtained was dried, water being separated as the pyridine azeotrope (2 ml.times.2).

[0055] iii) Coupling:

[0056] The dimer (DMTrOTpoC.sup.Bzpo-) and mesitylen sulfonylnitrothiazolide (MSNT) (80 mg) were dissolved in pyridine (0.5 ml). The solution was added into the reaction syringe with the polymer adduct I, and the mixture was shaked for 1 hour at room temperature. The reaction mixture was filtered through filter glass by nitrogen gas, and washed with pyridine (2 ml.times.3) to give the polymer adduct II.

[0057] iv) Acetylation of Unreacted 5'-hydroxy Groups:

[0058] To the polymer adduct II obtained as above, pyridine (0.9 ml) and acetic anhydride (0.1 ml) were added and the mixture was shaked for 15 minutes. Then the reaction solution was removed through filter glass and the resultant polymer was washed successively with pyridine (2 ml.times.3), methanol (2 ml.times.3) and dichloromethane (2 ml.times.3), and then dried by nitrogen gas stream. The polymer adduct (DMTrOTpoC.sup.BzpoTO-succinyl-polystyrene polymer) can use for the next coupling step.

[0059] (2) Synthesis of DMTrOTpoC.sup.BzpoC.sup.BzpoTpoC.sup.BzpoTO-succin- yl-polystyrene polymer:

[0060] DMTrOTpoC.sup.BzpoC.sup.BzpoTpoC.sup.BzpoTO-succinyl-polystyrene polymer was synthesized from DMTrOTpoC.sup.BzpoTO-succinyl-polystyrene polymer and DMTrOTpoC.sup.BzpoC.sup.BzpoCE (44.9 mg) according to similar conditions as above (1).

[0061] (3) Synthesis of DMTrOA.sup.BzpoG.sup.iBpoA.sup.BzpoTpoC.sup.BzpoC.- sup.BzpoTpo-C.sup.BzpoTO-succinyl-polystyrene polymer:

[0062] DMTrOA.sup.BzpoG.sup.iBpoA.sup.BzpoTpoC.sup.BzpoC.sup.BzpoTpoC.sup.- BzpoTO-succinyl-polystyrene polymer was synthesized from DMTrOTpoC.sup.BzpoC.sup.BzpoTpoC.sup.BzpoTO-succinyl-polystyrene polymer and DMTrOA.sup.BzpoG.sup.iBpoA.sup.BzpoCE (48.5 mg) according to similar conditions as above (1).

[0063] (4) Synthesis of DMTrOC.sup.BzpoG.sup.iBpoTpoA.sup.BzpoG.sup.iBpoA.- sup.BzpoTpo-C.sup.BzpoC.sup.BzpoTpoC.sup.BzpoTO-succinyl-polystyrene polymer:

[0064] DMTrOC.sup.BzpoG.sup.iBpoTpoA.sup.BzpoG.sup.iBpoA.sup.BzpoTpoC.sup.- BzpoC.sup.BzpoTpo-C.sup.BzpoTO-succinyl-polystyrene polymer was synthesized from DMTrOA.sup.BzpoG.sup.iBpoA.sup.BzpoTpoC.sup.BzpoC.sup.Bz- poTpoC.sup.BzpoTO-succinyl-polystyrene polymer and DMTrOC.sup.BzpoG.sup.iB- poTpoCE (45.1 mg) according to similar conditions as above (1).

[0065] (5) Synthesis of DMTrOC.sup.BzpoTpoG.sup.iBpoC.sup.BzpoG.sup.iBpoTp- oA.sup.Bzpo-G.sup.iBpoA.sup.BzpoTpoC.sup.BzpoC.sup.BzpoTpoC.sup.BzpoTO-suc- cinyl-polystyrene polymer:

[0066] DMTrOC.sup.BzpoTpoG.sup.iBpoC.sup.BzpoG.sup.iBpoTpoA.sup.BzpoG.sup.- iBpoA.sup.BzpoTpo-C.sup.BzpoC.sup.BzpoTpoC.sup.BzpoTO-succinyl-polystyrene polymer (60 mg) was synthesized from DMTrOC.sup.BzpoG.sup.iBpoTpoA.sup.Bz- poG.sup.iBpoA.sup.BzpoTpo-C.sup.BzpoC.sup.BzpoTpoC.sup.BzpoTO-succinyl-pol- ystyrene polymer and DMTrOC.sup.BzpoTpoG.sup.iBpoCE (45.1 mg) according to similar conditions as above (1). At this final step, unreacted 5'-hydroxy group was not necessary to protect with an acetyl group.

[0067] (6) Synthesis of HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH:

[0068] DMTrOC.sup.BzpoTpoG.sup.iBpoC.sup.BzpoG.sup.iBpoTpoA.sup.BzpoG.sup.- iBpoA.sup.BzpoTpo-C.sup.BzpoC.sup.BzpoTpoC.sup.BzpoTO-succinyl-polystyrene polymer (60 mg) was treated with 1M N,N,N',N'-tetramethyleneguanidium pyridine 2-aldoximate (in dioxane-water (1:1: v/v, 1 ml)) at 37.degree. C. for 20 hours in a sealed tube. To the reaction mixture 28% (w/w) aqueous ammonia (12 ml) was added, and the mixture was heated at 60.degree. C. for 5 hours. The solid polymer was removed by filtration and washed with water (10 ml). The filtrate and washed solution were evaporated to dryness, and the residue was treated with 80% aqueous acetic acid (25 ml) at room temperature for 15 minutes. After removal of the solvent, the residue was dissolved in 0.1M triethylammonium carbonate buffer (pH 7.5, 25 ml) was washed with diethylether (3.times.25 ml). Aqueous layer was evaporated to dryness and the residue was dissolved in 0.1M triethylammonium carbonate buffer (pH 7.5, 2 ml) to yield curde HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH in the solution.

[0069] (7) Purification of HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH

[0070] i) First purification of the crude product was performed by column chromatography on Biogel P2 (Biolad) (24.times.2.6 cm ID). The fractions corresponding to the first eluted peak (50 mM ammonium acetate containing 0.1 mM EDTA, flow rate: 1 ml/min) were collected and freeze-dried to give the first purified product.

[0071] ii) Second purification of the first purified product was performed by high performance liquid chromatography (HPLC) on CDR-10 (Mitsubishi Kasei) (25 cm.times.4.6 mm ID) using a linear gradient of 1M ammonium acetate-10% (v/v) aqueous ethanol to 4.5 M ammonium acetate-10% (v/v) aqueous ethanol (80 minutes, flow rate: 1 ml/minute, 60.degree. C.) to give the second purified product.

[0072] iii) Third purification of the second purified product was performed by reverse phase HPLC (Rp-18-5.mu.(.times.77) (Merck), 15 cm.times.4 mm ID) using a linear gradient of 0.1 M ammonium acetate to 0.1 M ammonium acetate-15% (v/v) aqueous acetonitrile (40 minutes, 1.5 ml/minute, room temperature) to give the final purified product.

[0073] (HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH)

[0074] (8) Analysis of oligonucleotide:

[0075] (HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH)

[0076] i) Digestion by phosphodiesterase

[0077] The mixture of HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH (10 .mu.g, 5.1 .mu.l), 0.2M MgCl.sub.2 (20 .mu.l), 0.2M tris-HCl (pH8.5) (20 .mu.l) and 0.1 mM EDTA (144.9 .mu.l) was treated with phosphodiesterase (10 unit, 10 .mu.l) at 37.degree. C. for 20 minutes, and then heated at 100.degree. C. for 2 minutes.

[0078] ii) Analysis by HPLC:

[0079] The oligonucleotide in the reaction mixture was analyzed by HPLC (CDR-10 (Mitsubishi Kasei), 25 cm.times.4.6 mm ID) using a linear gradient of water to 2.0 M ammonium acetate (pH 3.4) (40 minutes, flow rate: 1.5 ml/minute, 60.degree. C.). From each peak area observed, its nucleotide composition was determined comparing with area of a standard sample.

[0080] Calcd: pCOH 4.000, pAOH 2.000, pTOH 5.000, pGOH 3.000

[0081] Observed: pCOH 3.770, pAOH 2.026, pTOH 5.237, pGOH 2.968

EXAMPLE 2

[0082] Synthesis of oligonucleotide:

[0083] Following oligonucleotides were prepared in a similar manner to that described in Example 1.

1 (1) HOApGpCpTpTpGpApApGpTpTpGpApGpCpApTpGOH (AH1) (2) HOApApTpTpCpApTpGpCpTpCpApApCpTpTpCpAOH (AH2) (3) HOApApTpTpCpGpGpTpApTpGpGpGpCOH (AH3) (4) HOTpTpCpApCpCpGpCpCpCpApTpApCpCpGOH (AH4) (5) HOGpGpTpGpApApGpCpTpApApApTpCpTOH (AH5) (6) HOCpGpCpApGpApGpApTpTpTpApGpCOH (AH6) (7) HOApApGpCpApApGpApGpGpApTpCpTpAOH (AH8) (8) HOTpGpCpTpTpTpGpGpTpGpGpCpCpGpTOH (AH9) (9) HOTpCpCpApTpApCpGpGpCpCpApCpCpAOH (AH10) (10) HOApTpGpGpApCpCpGpCpApTpCpGpCpTOH (AH11) (11) HOTpGpApGpCpApCpCpGpApTpGpCpGpGOH (AH12) (12) HOGpCpTpCpApGpTpCpCpGpGpTpCpTpGOH (AH13) (13) HOCpApGpCpCpCpApGpApCpCpGpGpApCOH (AH14) (14) HOGpGpCpTpGpTpApApCpTpCpTpTpTpCOH (AH15) (15) HOTpApApCpGpGpApApApGpApGpTpTpAOH (AH16) (16) HOCpGpTpTpApCpTpGpApTpApGOH (AH17) (17) HOGpApTpCpCpTpApTpCpApGOH (AH18)

EXAMPLE 3

[0084] Synthesis of oligonucleotides:

[0085] Following oligonucleotides were prepared by a similar manner to that of Example 1.

2 (1) HOApApTpTpTpGpCpCpGpApCpAOH (A) (2) HOCpGpTpTpApTpGpApTpGpTpCpGpGpCpAOH (B) (3) HOTpCpApTpApApCpGpGpTpTpCpTpGpGpCOH (C) (4) HOGpApApTpApTpTpTpGpCpCpApGpApApCOH (D) (5) HOApApApTpApTpTpCpTpGpApApApTpGpAOH (E) (6) HOTpCpApApCpApGpCpTpCpApTpTpTpCpAOH (F) (7) HOGpCpTpGpTpTpGpApCpApApTpTpApApTOH (G) (8) HOGpTpTpCpGpApTpGpApTpTpApApTpTpGOH (H) (9) HOCpApTpCpGpApApCpTpApGpTpTpApApCOH (I) (10) HOGpCpGpTpApCpTpApGpTpTpApApCpTpAOH (J) (11) HOTpApGpTpApCpGpCpApApGpTpTpCpApCOH (K) (12) HOCpTpTpTpTpTpApCpGpTpGpApApCpTpTOH (L) (13) HOGpTpApApApApApGpGpGpTpApTOH (M') (14) HOCpGpApTpApCpCOH (N') (15) HOGpTpApApApApApGpGpGpTpApTpCpGOH (M) (16) HOApApTpTpCpGpApTpApCpCOH (N) (17) HOApApTpTpCpApTpGpGpCpTOH (SA) (18) HOGpGpTpTpGpTpApApGpApApCpTpTpCpTOH (SB) (19) HOTpTpTpGpGpApApGpApCpTpTpTOH (SC) (20) HOCpApCpTpTpCpGpTpGpTpTpGpApTpApGOH (SD) (21) HOTpTpApCpApApCpCpApGpCpCpApTpGOH (SE) (22) HOCpCpApApApApGpApApGpTpTpCOH (SF) (23) HOCpGpApApGpTpGpApApApGpTpCpTpTOH (SG) (24) HOGpApTpCpCpTpApTpCpApApCpAOH (SH)

EXAMPLE 4

[0086] Synthesis of oligonucleotides:

[0087] Following oligonucleotides were prepared by a similar manner to that of Example 1.

3 (1) HOApApCpTpApGpTpApCpGpCOH (Np1) (2) HOApApCpTpTpGpCpGpTpApCpTpApGpTpTOH (Np4) (3) HOApApGpTpTpCpApCpGpTpApApApApApGOH (Np2) (4) HOApTpApCpCpCpTpTpTpTpTpApCpGpTpGOH (Np5) (5) HOGpGpTpApTpCpGpApTpApApApApTpGOH (Np3) (6) HOGpTpApGpApApCpApTpTpTpTpApTpCpGOH (Np6) (7) HOTpTpCpTpApCpTpTpCpApApCpApApAOH (Cd1) (8) HOGpGpTpCpGpGpTpTpTpGpTpTpGpApAOH (Cd2) (9) HOCpCpGpApCpCpGpGpCpTpApTpGOH (Cd3) (10) HOGpCpTpGpGpApGpCpCpApTpApGpCpCOH (G2) (11) HOGpCpTpCpCpApGpCpTpCpTpCpGpTpCOH (H1) (12) HOCpGpGpTpGpCpGpCpGpApCpGpApGpAOH (H2) (13) HOGpCpGpCpApCpCpGpCpApGpApCpTpGOH (I1) (14) HOGpApTpApCpCpApGpTpCpTpGOH (Cd4) (15) HOGpTpApTpCpGpTpApGpApCpGOH (Cd5) (16) HOApCpCpCpTpCpGpTpCpTpApCOH (Cd6) (17) HOApGpGpGpTpGpGpCpGpApTpGOH (Cd7) (18) HOApApTpTpCpApTpCpGpCpCOH (Cd8)

EXAMPLE 5

[0088] Construction and Cloning of the Synthetic trp promoter II Gene (as Illustrated in FIGS. 6 and 7):

[0089] The trp promoter II gene was constructed by the similar method as described in Example 7 (as illustrated in FIG. 6). The synthetic gene was ligated with EcoRI-BamHI fragment of pBR322 (commercially available: Takarashuzo, NEB, etc.) and then E. coli HB101 (ATCC 33694) was transformed with the ligation product. The plasmid obtained from the transformant of .sup.RAmp and .sup.STet was digested with HpaI to confirm a band (4.1 kbp), and then digested with BamHI to confirm a band of 90 b.p. on PAGE. Moreover, the fragment of 56 b.p. by EcoRI-BamHI digestion was confirmed by the comparison with size marker on PAGE. This plasmid was named pTrpEB7 and used construction of expression vector.

EXAMPLE 6

[0090] Construction and Cloning of trp promoter vector (pBR322trp) (as Illustrated in FIG. 8):

[0091] Plasmid pBR322 (9 .mu.g) was digested with EcoRI and BamHI restriction endnucleases. Reaction was terminated by heating at 65.degree. C. for 5 minutes and the fragments were separated by electrophoresis on a 0.8% agarose gel to give the small fragment (500 ng) of 375 b.p. On the other hand, plasmid pTrpEB7 (10 .mu.g) was digested with EcoRI and BamHI, followed by preparative gel electrophoresis to give the large fragment (5 .mu.g) of 4094 b.p. The pTrpEB7 EcoRI-BamHI fragment (4094 b.p., 200 .mu.g) was ligated with the pBR322 EcoRI-BamHI fragment (375 b.p., 100 ng) in the ligation buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl.sub.2, 20 mM DTT, 1 mM ATP, 1 mM spermidine, 50 .mu.g/ml BSA) (20 .mu.l) containing T4 DNA ligase. (Takarashuzo: 360 unit) at 15.degree. C. overnight. The ligated mixture was transformed into E. coli HB101 by Kushiner's method (Cf. T. Maniatis et al Molecular Cloning p252 (1982), Cold Spring Harbor Laboratory) and tetracycline resistant transformants were obtained on the plate containing tetracycline (25 .mu.g/ml). The plasmid pBR322trp isolated from the transformant was digested with EcoRI-BamHI (375 b.p., 4094 b.p.) and HpaI (4469 b.p.) to confirm the trp promoter gene by 7.5% PAGE and 0.8% agarose gel electrophoresis.

EXAMPLE 7

[0092] Construction of the Synthetic trp promoter III Gene (as Illustrated in FIG. 9):

[0093] Each oligonucleotides (B-M') (each 0.2 n mole) of block I', II' and III' were phosphorylated with T4 polynucleotide kinase (BRL; 2.5 unit) in the ligation buffer (70 .mu.l) at 37.degree. C. for 1 hour. To the reaction mixture of each blocks T4 DNA ligase (300 unit) and 20 mM ATP (2 .mu.l) were added, and the mixture was incubated at 15.degree. C. for 30 minutes. The reaction was terminated by heating at 65.degree. C. for 10 minutes. The reaction mixture of these blocks (I', II' and III') was put together and mixed with unphosphorylated oligonucleoties (A, N') in the presence of T4 DNA ligase (360 unit) and 20 mM ATP (2 .mu.l). After the incubation of the mixture at 15.degree. C. for 1 hour, the last ligation product was purified by 2-16% gradient polyacrylamide gel electrophoresis (PAGE) to give the 106 b.p. synthetic trp promoter III gene.

EXAMPLE 8

[0094] Construction and Cloning of Double trp promoter vector (p322dtrpS)(as Illustrated in FIG. 10):

[0095] Plasmid pBR322trp was digested with EcoRI and ClaI, followed by preparative agarose gel electrophoresis to give the large fragment of 4446 b.p. This fragment (4446 b.p.) was ligated with trp promoter III gene (106 b.p.) obtained in Example 7 in the presence of T4 DNA ligase. The ligated mixture was transformed into E. coli HB101 to give the transformants of ampicillin and tetracycline resistance. The plasmid p322dtrpS obtained from the transformant was confirmed by restriction endonuclease analysis ClaI-BamHI (352 b.p.), HpaI (107 b.p.) and AatII-ClaI (287 b.p.).

EXAMPLE 9

[0096] Construction of Peptide Cd Gene With a Part of DNA Fragment of Synthetic trp promoter III (as Illustrated in FIGS. 11 and 12):

[0097] Each oligonucleotides (0.2 n mole) (Np1-Cd8, shown in Example 4) of block I", II" and III" were phosphorylated with T4 polynucleotide kinase (2.5 unit) in ligation buffer (60 .mu.l) at 37.degree. C. for 1 hour. To the reaction mixture of each block T4 DNA ligase (360 unit) and ATP (2 .mu.l) was added, the mixture was incubated at 15.degree. C. for 1 hour. The reaction mixture of these blocks (I", II" and III") was put together and incubated with T4 DNA ligase (360 unit) and 20 mM ATP (2 .mu.l) at 15.degree. C. overnight, and then heated at 80.degree. C. for 10 minutes. To the mixture 500 mM NaCl (20 .mu.l) and EcoRI (20 unit) were added. After the incubation at 37.degree. C. for 2 hours, the last ligation product was purified by 15% PAGE to give the peptide Cd gene with a part of DNA fragment of synthetic trp promoter III (125 b.p.), DNA sequence of which is illustrated in FIG. 12.

EXAMPLE 10

[0098] Construction and Cloning of plasmid pCd.gamma. (as Illustrated in FIG. 13):

[0099] Plasmid p.gamma.trp(4544 b.p.) (Cf. GB2164650A published on Mar. 26, 1986; Escherichia coli F-9 containing this plasmid p.gamma.trp has been depositing with. FRI(Japan) under the number FERM BP-905 from Sep. 20, 1984) was digested with HpaI and EcoRI to give a large fragment (4510 b.p.), which was ligated with the peptide Cd gene with a part of DNA fragment of synthetic trp promoter III (125 b.p.) as obtained in Example 9 in the presence of T4 DNA ligase. The ligated mixture was transformed into E. coli HB101. The plasmid (pCd.gamma.) obtained from the transformant of .sup.RAmp was confirmed by restriction endonuclease analysis:

[0100] ClaI-BamHI (543 b.p.), ClaI-HindIII (273 b.p.), ClaI-EcoRI (93 b.p.) and AatII-ClaI (180 b.p.).

EXAMPLE 11

[0101] Construction and Cloning of plasmid pCd.gamma.trpSd (as Illustrated in FIG. 14):

[0102] The plasmid pCd.gamma. was digested with ClaI and BamHI to give the smaller fragment (543 b.p.), which was ligated with the ClaI-BamHI fragment (4223 b.p.) of p322dtrpS (Example 7) in the presence of T4 DNA ligase. The ligated mixture was transformed into E. coli HB101. The plasmid (pCd.gamma.trpSd) obtained from the transformant of .sup.RAmp was confirmed by restriction endonuclease analysis:

[0103] HpaI-BamHI (107,575 b.p.), ClaI-BamHI (543 b.p.),

[0104] PstI-EcoRI (1057 b.p.), EcoRI-BamHI (450 b.p.)

[0105] HindIII-BamHI (270 b.p.), ClaI-HindIII (273 b.p.)

EXAMPLE 12

[0106] Preparation of .alpha.-hANP Gene With Linker DNA (as Illustrated in FIG. 15):

[0107] Each oligonucleotides (AH2-AH17) (each 0.2 n mole) of block I and II were phosphorylated with T4 polynucleotide kinase (2.5 unit) in the ligation buffer (70 .mu.l) at 37.degree. C. for 1 hour. To the reaction mixture of each blocks T4 DNA ligase (300 unit) and 20 mM ATP (2 .mu.l) were added, and the mixture was incubated at 15.degree. C. for 30 minutes. The reaction was terminated by heating at 65.degree. C. for 10 minutes. The reaction mixture of two blocks (I'" and II'") was put together and mixed with unphosphorylated oligonucleotides (AH1, AH18) in the presence of T4 DNA ligase (300 unit) and 20 mM ATP (2 .mu.l). After the incubation of the mixture at 15.degree. C. for 1 hour, the last ligation product was purified by 2-16% gradient PAGE to give the 134 b.p. .alpha.-hANP gene with linker DNA (as illustrated in FIG. 5).

EXAMPLE 13

[0108] Construction and Cloning of .alpha.-hANP Expression Vector pCLaHtrpSd (as Illustrated in FIG. 16):

[0109] The plasmid pCd.gamma.trpSd was digested with HindIII and BamHI to give the larger fragment (4743 b.p.), which was ligated with the .alpha.-hANP gene with linker DNA (134 b.p.) in the presence of T4 DNA ligase. The ligated mixture was transformed into E. coli HB101 to give a transformant H1. The plasmid (pCLaHtrpSd) (which contains CLaH protein(peptide CLa-fused .alpha.-hANP protein)gene, DNA sequence of which is illustrated in FIG. 17) obtained from the transformant of .sup.RAmp (E. coli H1) was confirmed by restriction endonuclease analysis:

[0110] AatII-ClaI (287 b.p.), ClaI-BamHI (407 b.p.),

[0111] ClaI-EcoRI (93, 198 b.p.), EcoRI-BamHI (116, 198 b.p.), HindIII-BamHI (134 b.p.), HpaI-BamHI (107, 439 b.p.).

EXAMPLE 14

[0112] Expression of a Gene Coding for the peptide CLa-Fused .alpha.-hANP (CLaH Protein):

[0113] An overnight culture of E. coli H1 containing the expression vector, plasmid pCLaHtrpSd in L broth (20 ml) containing 50 .mu.g/ml ampicillin was diluted in M9 medium (400 ml) containing 0.2% glucose, 0.5% casamino acid (acid-hydrolyzed casein), 50 .mu.g/ml vitamin B1 and 25 .mu.l/ml ampicillin, and the E. coli was cultured at 37.degree. C. When A600 (absorbance at 600 nm) of the cultured broth was 0.5, .beta.-indole acrylic acid (2 mg/ml ethanol; 2 ml) was added and the cells were incubated for 3 hours (final A600=1.85). Then the cells were harvested by centrifugation (6000 rpm, 4.degree. C., 5 minutes).

EXAMPLE 15

[0114] Isolation and Purification of .alpha.-hANP:

[0115] (1) Isolation and Purification of the peptide CLa-Fused .alpha.-hANP (CLaH Protein)

[0116] The wet cell paste from the cultured broth (600 ml) as prepared in Example 14 was suspended in 8 ml of 10 mM PBS-EDTA (pH 7.4) (NaCl (8.0 g), KCl (0.2 g), Na.sub.2HPO4 12H.sub.2O (2.9 g), KH.sub.2PO.sub.4 (0.2 g), EDTA (3.73 g)/liter) and cells were destroyed by sonication at 0.degree. C. The pellet was collected by centrifugation at 15,000 rpm for 20 minutes (4.degree. C.), and suspended in 8 ml of 6M guanidine-HCl, 10 mM PBS-EDTA and 2 mM .beta.-mercaptoethanol and the suspension was treated by super sonication at 0.degree. C. The suspension was centrifuged at 15,000 rpm for 20 minutes (4.degree. C.) and the supernatant was dialyzed overnight at 4.degree. C. against 10 mM pBS-EDTA solution containing p-nitrophenyl methylsulfonyl fluoride (PMSF). After the fraction dialyzed was centrifuged (15,000 rpm, 4.degree. C., 20 minites), the pellet was dissolved in 100 mM Tris-HCl buffer (pH 8.0) (8 ml) containing 6M guanidine-HCl, 10 mM EDTA and 100 mM dithiothreitol and the solution was stood overnight. The solution was dialyzed against 1M acetic acid (0.5 liters) containing 10 mM 2-mercaptoethanol twice and adjusted to pH 8.0 with trisaminomethane. The resulting precipitate (fused protein; 15.2 mg) was collected by centrifugation (3.000 rpm, 10 minutes), and washed with 10 mM sodium acetate buffer (pH 5.0).

[0117] (2) Elimination of peptide CLa from the peptide CLa Fused .alpha.-hANP with Achromobacter protease I(API):

[0118] The fused protein obtained above was suspended in 10 mM sodium acetate buffer (pH 5.0) (30 ml) containing 8M urea, the suspension was incubated with Achromobactor protease I(API) (0.25 unit) (Wako pure chemical industries, Ltd) at 37.degree. C. for 2 hours. The reaction mixture was diluted with distilled water (30 ml), adjusted to pH 9.0 with trisaminomethane, and then incubated with additional API (0.25 unit) at 37.degree. C. for 2 hours. The reaction solution was diluted with 10 mM sodium phosphate buffer (pH 7.0) (120 ml), and adjusted to pH 7 with acetic acid. The solution was applied to a Sp-sephadex C-25 column (15 ml) equilibrated with 10 mM solium phosphate buffer (pH 7.0). The column was washed with the same buffer, and eluted with 10 mM sodium phosphate buffer (pH 8.0) containing 0.5M aqueous sodium chloride to collect the fractions containing a partial purified .alpha.-hANP (0.4 mg).

[0119] (3) High Performance Liquid Chromatography (HPLC):

[0120] The pooled fraction obtained in the above (2) was concentrated in vacuo, dialyzed against water (300 ml), and purified by reverse phase HPLC to give a pure .alpha.-hANP (0.3 mg).

4 HPLC condition (preparation) column: Beckman Ultrapore semi-prep. (.phi.10 .times. 250 mm) flow rate: 2.5 ml/minute elution: linear gradient from 10% to 60% acetonitrile in 0.01 M trifluoroacetic acid over 50 minutes. monitor absorbance at 214 nm (analysis) column: Beckmann Ultrapore RPSC (.phi. 4.6 .times. 75 mm) flow rate: 1 ml/minute elution: same condition as the preparation retention time: 11.9 minutes

[0121] The .alpha.-hANP was supperimposed with authentic .alpha.-hANP (sold by Funakoshi)

[0122] (4) Amino Acid Analysis of .alpha.-hANP

[0123] The sample was reduced and carboxymethylated, and then hydrolyzed with 6N HCl at 110.degree. C. for 24 hours. The amino acid composition of .alpha.-hANP was obtained using a Waters amino acid analysis system.

[0124] Amino acid compositions (residues per mole) of .alpha.-hANP were coincided with the expected values.

[0125] (5) Amino Acid Sequence Analysis of .alpha.-hANP

[0126] The N-terminal amino acid sequence of .alpha.-hANP was determined by Edman's method (DABITC method) [described in FEBS Lett., 93,205 (1978)] to confirm N-terminal Ser and Leu sequence. C-terminal amino acids (Ser-Phe-Arg-Tyr) were determined by the digestion with carboxypeptidase and the followed amino acid analysis using a Waters amino acid analysis system. The whole amino acid sequence of .alpha.-hANP obtain in the above Example was determined by using both procedures and was identical with the known sequence of .alpha.-hANP.

EXAMPLE 16

[0127] Construction and Cloning of plasmid pBR322trpSs (as Illustrated in FIG. 18):

[0128] Plasmid pBR322 was digested with EcoRI and ClaI. The large fragment (4340 bp) was purified by 0.8% agarose gel electrophoresis, and ligated to the synthetic trp promoter III gene in the presence of T4 DNA ligase and 1 mM ATP. The ligation mixture was used to transform E. coli HB101. The plasmid DNA (pBR322trpSs) was isolated from a transformed clone .sup.RAmp) and charactarized by restriction endonuclease analysis.

[0129] Analysis data: Hpa I; 4445 bp, ClaI-Pst I; 834 bp

EXAMPLE 17

[0130] Construction and Cloning of plasmid pCLaHtrp-2 (as Illustrated in FIG. 19):

[0131] Plasmid pCLaHtrpSd was digested with ClaI and BamHI. The small fragment (407 bp) was isolated. On the other hand pBR322trpSs was digested with ClaI and BamHI. The larger fragment (4093 bp) was isolated and ligated to the former DNA (407 bp). After transformation of E. coli HB101 with the ligation mixture, the desired plasmid (pCLaHtrp-2) was isolated from a transformed clone(.sup.RAmp) and characterized by restriction enzyme analysis: ClaI-Pst I; 834 bp, ClaI-BamHI; 407 bp

EXAMPLE 18

[0132] Synthesis of oligonucleotides:

[0133] Following oligonucleotides were prepared in a similar manner to that of Example 1.

5 (1) HOGpApTpCpCpTpCpGpApGpApTpCpApAOH (T1) (2) HOGpCpCpTpTpTpApApTpTpGpApTpCpTpCpGpApGOH (T2) (3) HOTpTpApApApGpGpCpTpCpCpTpTpTpTpGpGpAOH (T3) (4) HOApApApApApGpGpCpTpCpCpApApApApGpGpAOH (T4) (5) HOGpCpCpTpTpTpTpTpTpTpTpTpTpGOH (T5) (6) HOTpCpGpApCpApApApApAOH (T6)

EXAMPLE 19

[0134] Construction and Cloning of Synthetic fd phage Terminator (as Illustrated in FIGS. 20 and 21):

[0135] The synthetic fd phage terminator was constructed by a similar method as described in Example 7 (as illustrated in FIG. 20).

[0136] Namely, DNA oligomers T2, T3, T4 and T5 (each 0.4 nmole) were mixed and phosphorylated with T4 polynucleotide kinase in the presence of 1 mM ATP. The reaction mixture was heated at 65.degree. C. for 10 minutes to inactivate the enzyme. To the resultant mixture, DNA oligomer T1 and T6 (each 0.8 nmole) and T4 DNA ligase were added. The mixture was incubated at 15.degree. C. for 30 minutes, and applied to 2.fwdarw.16% gradient polyacrylamide gel electrophoresis. The desired DNA fragment (47 bp) was recovered by electroelution and ligated to the larger fragment of pBR322 digested with BamHI and Sal I (4088 bp). After transformation of E. coli HB101 with the ligation mixture, the desired plasmid (pter) was isolated from a transformed clone (.sup.RAmp).

[0137] Restriction enzyme analysis: BamHI-Sal I; 47 bp, Ava I; 817 bp

EXAMPLE 20

[0138] Construction and Cloning of .alpha.-hANP expression vector plasmid pCLaHtrp3t (as Illustrated in FIG. 22):

[0139] Plasmid pCLaHtrp-2 was digested with Pst I and BamHI. From the digestion mixture, the small fragment (1241 bp) was isolated and ligated to the large fragment of pter 21 obtained from digestion of pter 21 with Pst I and BamHI (3005 bp).

[0140] The ligation mixture was transformed into E. coli HB101 to give a transformant E. coli H2. The plasmid CLaHtrp3t (which contains CLaH protein gene) obtained from the transformant of .sup.RAmp (E. coli H2) was confirmed by restriction endonuclease analysis: ClaI-EcoRI; 93 bp, 198 bp, HindIII-BamHI; 134 bp, PstI-ClaI-XhoI; 834 bp, 411 bp

EXAMPLE 21

[0141] Production of .alpha.-hANP Using E. coli H2:

[0142] .alpha.-hANP was obtained in a similar manner to those of Example 14 and 15 using E. coli H2 in place of E. coli H1.

[0143] Amino acid sequence of thus obtained .alpha.-hANP was identical with the known sequence of .alpha.-hANP.

Sequence CWU 1

1

88 1 28 PRT Homo sapiens 1 Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly 1 5 10 15 Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 20 25 2 15 DNA Artificial Sequence synthetic DNA 2 ctgcgtagat cctct 15 3 18 DNA Artificial Sequence synthetic DNA 3 agcyygaagy ygagcayg 18 4 18 DNA Artificial Sequence synthetic DNA 4 aattcatgct caacttca 18 5 14 DNA Artificial Sequence synthetic DNA 5 aattcggtat gggc 14 6 16 DNA Artificial Sequence synthetic DNA 6 ttcaccgccc ataccg 16 7 15 DNA Artificial Sequence synthetic DNA 7 ggtgaagcta aatct 15 8 14 DNA Artificial Sequence synthetic DNA 8 cgcagagatt tagc 14 9 15 DNA Artificial Sequence synthetic DNA 9 aagcaagagg atcta 15 10 15 DNA Artificial Sequence synthetic DNA 10 tgctttggtg gccgt 15 11 15 DNA Artificial Sequence synthetic DNA 11 tccatacggc cacca 15 12 15 DNA Artificial Sequence synthetic DNA 12 atggaccgca tcgct 15 13 15 DNA Artificial Sequence synthetic DNA 13 tgagcaccga tgcgg 15 14 15 DNA Artificial Sequence synthetic DNA 14 gctcagtccg gtctg 15 15 15 DNA Artificial Sequence synthetic DNA 15 cagcccagac cggac 15 16 15 DNA Artificial Sequence synthetic DNA 16 ggctgtaact ctttc 15 17 15 DNA Artificial Sequence synthetic DNA 17 taacggaaag agtta 15 18 12 DNA Artificial Sequence synthetic DNA 18 cgttactgat ag 12 19 11 DNA Artificial Sequence synthetic DNA 19 gatcctatca g 11 20 12 DNA Artificial Sequence synthetic DNA 20 aatttgccga ca 12 21 16 DNA Artificial Sequence synthetic DNA 21 cgttatgatg tcggca 16 22 16 DNA Artificial Sequence synthetic DNA 22 tcataacggt tctggc 16 23 16 DNA Artificial Sequence synthetic DNA 23 gaatatttgc cagaac 16 24 16 DNA Artificial Sequence synthetic DNA 24 aaatattctg aaatga 16 25 16 DNA Artificial Sequence synthetic DNA 25 tcaacagctc atttca 16 26 16 DNA Artificial Sequence synthetic DNA 26 gctgttgaca attaat 16 27 16 DNA Artificial Sequence synthetic DNA 27 gttcgatgat taattg 16 28 16 DNA Artificial Sequence synthetic DNA 28 catcgaacta gttaac 16 29 16 DNA Artificial Sequence synthetic DNA 29 gcgtactagt taacta 16 30 16 DNA Artificial Sequence synthetic DNA 30 tagtacgcaa gttcac 16 31 15 DNA Artificial Sequence synthetic DNA 31 cttttacgtg aactt 15 32 13 DNA Artificial Sequence synthetic DNA 32 gtaaaaaggg tat 13 33 7 DNA Artificial Sequence synthetic DNA 33 cgatacc 7 34 15 DNA Artificial Sequence synthetic DNA 34 gtaaaaaggg tatcg 15 35 11 DNA Artificial Sequence synthetic DNA 35 aattcgatac c 11 36 11 DNA Artificial Sequence synthetic DNA 36 aattcatggc t 11 37 16 DNA Artificial Sequence synthetic DNA 37 ggttgtaaga acttct 16 38 13 DNA Artificial Sequence synthetic DNA 38 tttggaagac ttt 13 39 16 DNA Artificial Sequence synthetic DNA 39 cacttcgtgt tgatag 16 40 15 DNA Artificial Sequence synthetic DNA 40 ttacaaccag ccatg 15 41 13 DNA Artificial Sequence synthetic DNA 41 ccaaaagaag ttc 13 42 15 DNA Artificial Sequence synthetic DNA 42 cgaagtgaaa gtctt 15 43 13 DNA Artificial Sequence synthetic DNA 43 gatcctatca aca 13 44 11 DNA Artificial Sequence synthetic DNA 44 aactagtacg c 11 45 16 DNA Artificial Sequence synthetic DNA 45 aacttgcgta ctagtt 16 46 16 DNA Artificial Sequence synthetic DNA 46 aagttcacgt aaaaag 16 47 16 DNA Artificial Sequence synthetic DNA 47 ataccctttt tacgtg 16 48 15 DNA Artificial Sequence synthetic DNA 48 ggtatcgata aaatg 15 49 16 DNA Artificial Sequence synthetic DNA 49 gtagaacatt ttatcg 16 50 15 DNA Artificial Sequence synthetic DNA 50 ttctacttca acaaa 15 51 15 DNA Artificial Sequence synthetic DNA 51 ggtcggtttg ttgaa 15 52 13 DNA Artificial Sequence synthetic DNA 52 ccgaccggct atg 13 53 15 DNA Artificial Sequence synthetic DNA 53 gctggagcca tagcc 15 54 15 DNA Artificial Sequence synthetic DNA 54 gctccagctc tcgtc 15 55 15 DNA Artificial Sequence synthetic DNA 55 cggtgcgcga cgaga 15 56 15 DNA Artificial Sequence synthetic DNA 56 gcgcaccgca gactg 15 57 12 DNA Artificial Sequence synthetic DNA 57 gataccagtc tg 12 58 12 DNA Artificial Sequence synthetic DNA 58 gtatcgtaga cg 12 59 12 DNA Artificial Sequence synthetic DNA 59 accctcgtct ac 12 60 12 DNA Artificial Sequence synthetic DNA 60 agggtggcga tg 12 61 11 DNA Artificial Sequence synthetic DNA 61 aattcatcgc c 11 62 15 DNA Artificial Sequence synthetic DNA 62 gatcctcgag atcaa 15 63 19 DNA Artificial Sequence synthetic DNA 63 gcctttaatt gatctcgag 19 64 18 DNA Artificial Sequence synthetic DNA 64 ttaaaggctc cttttgga 18 65 18 DNA Artificial Sequence synthetic DNA 65 aaaaaggctc caaaagga 18 66 13 DNA Artificial Sequence synthetic DNA 66 gccttttttt ttt 13 67 10 DNA Artificial Sequence synthetic DNA 67 tcgacaaaaa 10 68 106 DNA Artificial Sequence synthetic trp promoter 68 aattgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc 60 atcgaactag ttaactagta cgcaagttca cgtaaaaagg gtatcg 106 69 164 DNA Artificial Sequence synthetic trp promoter 69 aatttgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat 60 catcgaacta gttaactagt aacgcaagtt cacgtaaaaa gggtatcgaa ttcatggctg 120 gttgtaagaa cttcttttgg aagactttca cttcgtgttg atag 164 70 105 DNA Artificial Sequence synthetic trp promoter 70 aatttgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat 60 catcgaacta gttaactagt acgcaagttc acgtaaaaag ggtat 105 71 308 DNA Homo sapiens 71 atgttctact tcaacaaacc gaccggctat ggctccagct ctcgtcgcgc accgcagact 60 ggtatcgtag acgagggtgg cgatgaattc atgtgttact gccaggaccc atatgtaaaa 120 gaagcagaaa accttaagaa atactttaat gcaggtcatt cagatgtagc ggataatgga 180 actcttttct taggcatttt gaagaattgg aaagaggaga gtgacagaaa aataatgcag 240 agccaaattg tctccttcta cttcaagctt gaagttgagc atgaattcgg tatgggcggt 300 gaagctaa 308 72 103 PRT Homo sapiens 72 Met Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15 Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 25 30 Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 40 45 Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 55 60 Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Asp Lys Ile Met Gln 65 70 75 80 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Gly His Gly Phe 85 90 95 Gly Met Gly Gly Glu Ala Lys 100 73 134 DNA Homo sapiens 73 agcttgaagt tgagcatgaa ttcggtatgg gcggtgaagc taaatctgtg cgtagatcct 60 cttgctttgg tggccgtatg gaccgcatcg gtgctcagtc cggtctgggc tgtaactctt 120 tccgttactg atag 134 74 42 PRT Homo sapiens 74 Lys Leu Glu Val Glu His Glu Phe Gly Met Gly Gly Glu Ala Lys Ser 1 5 10 15 Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Gly Ala Gln 20 25 30 Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 35 40 75 4 PRT Homo sapiens 75 Ser Phe Arg Tyr 1 76 28 PRT Homo sapiens 76 Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly 1 5 10 15 Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 20 25 77 84 DNA Homo sapiens 77 tctctgcgta gatcctcttg ctttggtggc cgtatggacc gcatcggtgc tcagtccggt 60 ctggggtgta actctttccg ttac 84 78 111 DNA Artificial Sequence synthetic DNA 78 aatttgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat 60 catcgaacta gttaactagt acgcaagttc acgtaaaaag ggtatcgaag g 111 79 167 DNA Artificial Sequence synthetic DNA 79 aatttgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat 60 catcgaacta gttaactagt acgcaagttc acgtaaaaag ggtatcgaat tcatggctgg 120 ttgtaagaac ttcttttgga agactttcac ttcgtgttga taggatc 167 80 107 DNA Artificial Sequence synthetic DNA 80 aatttgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat 60 catcgaacta gttaactagt acgcaagttc acgtaaaaag ggtatcg 107 81 309 DNA Homo sapiens CDS (1)..(309) 81 atg ttc tac ttc aac aaa ccg acc ggc tat ggc tcc agc tct cgt cgc 48 Met Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15 gca ccg cag act ggt atc gta gac gag ggt ggc gat gaa ttc atg tgt 96 Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 25 30 tac tgc cag gac cca tat gta aaa gaa gca gaa aac ctt aag aaa tac 144 Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 40 45 ttt aat gca ggt cat tca gat gta gcg gat aat gga act ctt ttc tta 192 Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 55 60 ggc att ttg aag aat tgg aaa gag gag agt gac aga aaa ata atg cag 240 Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln 65 70 75 80 agc caa att gtc tcc ttc tac ttc aag ctt gaa gtt gag cat gaa ttc 288 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Glu His Glu Phe 85 90 95 ggt atg ggc ggt gaa gct aaa 309 Gly Met Gly Gly Glu Ala Lys 100 82 103 PRT Homo sapiens 82 Met Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15 Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 25 30 Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 40 45 Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 55 60 Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln 65 70 75 80 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Glu His Glu Phe 85 90 95 Gly Met Gly Gly Glu Ala Lys 100 83 138 DNA Homo sapiens CDS (3)..(131) 83 ag ctt gaa gtt gag cat gaa ttc ggt atg ggc ggt gaa gct aaa tct 47 Leu Glu Val Glu His Glu Phe Gly Met Gly Gly Glu Ala Lys Ser 1 5 10 15 ctg cgt aga tcc tct tgc ttt ggt ggc cgt atg gac cgc atc ggt gct 95 Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala 20 25 30 cag tcc ggt ctg ggc tgt aac tct ttc cgt tac tga taggatc 138 Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 35 40 84 42 PRT Homo sapiens 84 Leu Glu Val Glu His Glu Phe Gly Met Gly Gly Glu Ala Lys Ser Leu 1 5 10 15 Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln 20 25 30 Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 35 40 85 128 DNA Artificial Sequence synthetic DNA and Homo sapien hybrid 85 aactagtacg caagttcacg taaaaagggt atcgataaa atg ttc tac ttc aac 54 Met Phe Tyr Phe Asn 1 5 aaa ccg acc ggc tat ggc tcc agc tct cgt cgc gca ccg cag act ggt 102 Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly 10 15 20 atc gta gac gag ggt ggc gat gaa gg 128 Ile Val Asp Glu Gly Gly Asp Glu 25 86 29 PRT Artificial Sequence synthetic DNA and Homo sapien hybrid 86 Met Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15 Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu 20 25 87 393 DNA Homo sapiens CDS (1)..(393) 87 atg ttc tac ttc aac aaa ccg acc ggc tat ggc tcc agc tct cgt cgc 48 Met Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15 gca ccg cag act ggt atc gta gac gag ggt ggc gat gaa ttc atg tgt 96 Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 25 30 tac tgc cag gac cca tat gta aaa gaa gca gaa aac ctt aag aaa tac 144 Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 40 45 ttt aat gca ggt cat tca gat gta gcg gat aat gga act ctt ttc tta 192 Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 55 60 ggc att ttg aag aat tgg aaa gag gag agt gac aga aaa ata atg cag 240 Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln 65 70 75 80 agc caa att gtc tcc ttc tac ttc aag ctt gaa gtt gag cat gaa ttc 288 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Glu His Glu Phe 85 90 95 ggt atg ggc ggt gaa gct aaa tct ctg cgt aga tcc tct tgc ttt ggt 336 Gly Met Gly Gly Glu Ala Lys Ser Leu Arg Arg Ser Ser Cys Phe Gly 100 105 110 ggc cgt atg gac cgc atc ggt gct cag tcc ggt ctg ggc tgt aac tct 384 Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser 115 120 125 ttc cgt tac 393 Phe Arg Tyr 130 88 131 PRT Homo sapiens 88 Met Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15 Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 25 30 Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 40 45 Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 55 60 Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln 65 70 75 80 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Glu His Glu Phe 85 90 95 Gly Met Gly Gly Glu Ala Lys Ser Leu Arg Arg Ser

Ser Cys Phe Gly 100 105 110 Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser 115 120 125 Phe Arg Tyr 130

Claims

We claim:

1. A process for the production of .alpha.-hANP by (1) culturing a microorganism transformed with an expression vector comprising a synthetic gene encoding an amino acid sequence of a protective peptide-fused .alpha.-hANP in a nutrient medium, (2) recovering the protective peptide-fused .alpha.-hANP from the cultured broth and (3) removing the protective peptide portion of the protective peptide-fused .alpha.-hANP.

2. A process for the production of .alpha.-hANP of claim 1, in which the microorganism is bacteria.

3. A process for the production of .alpha.-hANP of claim 2, in which the bacteria is a strain belonging to the genus Escherichia.

4. A process for the production of .alpha.-hANP of claim 3, in which the strain is Escherichia coli.

5. A process for the production of .alpha.-hANP of claim 1, in which .alpha.-hANP gene portion of the synthetic gene is represented by the following DNA sequence:

6 Coding: 5'-TCT CTG CGT AGA TCC TCT TGC TTT GGT Noncoding: 3'-AGA GAC GCA TCT AGG AGA ACG AAA CCA GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC GGC TGT AAC TCT TTC CGT TAC-3' CCG ACA TTG AGA AAG GCA ATG-5'

6. A process for the production of .alpha.-hANP of claim 1, in which the protective peptide-fused .alpha.-hANP has the amino acid sequence represented in FIG. 17.

7. A process for the production of .alpha.-hANP of claim 1, the protectice peptide portion of protective peptide-fused .alpha.-hANP is removed in the presence of API.

8. A chemically synthsized gene encoding amino acid sequence of .alpha.-hANP: H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-I- le-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH

9. The chemically synthsized gene of claim 8, which is represented by the following DNA sequence:

7 Coding: 5'-TCT CTG CGT AGA TCC TCT TGC TTT GGT Noncoding: 3'-AGA GAC GCA TCT AGG AGA ACG AAA CCA GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC GGC TGT AAC TCT TTC CGT TAC-3' CCG ACA TTG AGA AAG GCA ATG-5'

10. A chemically synthesized protective peptide gene having DNA sequence encoding lysine as C-terminal of the protective peptide.

11. The chemically synthesized protective peptide gene of claim 10, which is represented by the DNA sequence of FIG. 4.

12. A recombinant vector comprising chemically synthesized .alpha.-hANP gene.

13. A recombinant vector of claim 13, in which the .alpha.-hANP gene is represented by the following DNA sequence:

8 Coding: 5'-TCT CTG CGT AGA TCC TCT TGC TTT GGT Noncoding: 3'-AGA GAC GCA TCT AGG AGA ACG AAA CCA GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC GGC TGT AAC TCT TTC CGT TAC-3' CCG ACA TTG AGA AAG GCA ATG-5'

14. A recombinant vector comprising chemically synthesized gene represented by the DNA sequence of FIG. 17.

15. A recombinant vector comprising chemically synthesized gene represented by the DNA sequence of FIG. 4.

16. A transformant comprising expression vector of chemically synthesized .alpha.-hANP gene.

17. A transformant of claim 16, in which the .alpha.-hANP gene is presented by the following DNA sequence:

9 Coding: 5'-TCT CTG CGT AGA TCC TCT TGC TTT GGT Noncoding: 3'-AGA GAC GCA TCT AGG AGA ACG AAA CCA GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC GGC TGT AAC TCT TTC CGT TAC-3' CCG ACA TTG AGA AAG GCA ATG-5'

18. A cleaving method of the fused-protein composed of a peptides having a lysine between a protective peptide and a targent peptide in the presence of API.

19. Synthetic trp promoter III represented by the DNA sequence of FIG. 3.