KR970009354B1 - Motilin-like polypeptide and use thereof - Google Patents

Abstract

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Description

Motirin-type polypeptides and uses thereof

1 is an explanatory diagram showing a step of inserting one motirin-type gene into a plasmid.

2 is an explanatory diagram showing a step of inserting two motirin-type genes arranged in tandem into a plasmid.

Figure 3 shows the magnificent contraction activity of the motirin-type polypeptide prepared by the method of the present invention and the pure [leucine ¹³ ] -motirin obtained by the conventional method when the contractile activity of ^10-6 M acetylcholine is 100%. Graph showing the results measured by.

Figure 4A is a chart showing the change of jangpan movement measured by the balun method using an unconscious living dog when controlling [leucine ¹³ ] -motirin-homoserine according to the present invention.

FIG. 4B is similar to FIG. 4A, and is a chart in the case where the conventional pure [leucine ¹³ ] -motirin is adjusted.

FIG. 5 shows the conventional leucine ^13- motirin and fold contractile activity measured by the balun method at various concentrations when the contractile activity of the prostaglandin F _2a agent of 30 μg / kg / hour is 100%. , Graph showing the results for [Leucine ¹³ ] -motirin-homoserine and salts thereof.

The present invention relates to a motinin type polypeptide, ie, a polypeptide having a motirin type pharmacological activity, a salt thereof, a method for preparing the polypeptide, and a use of the polypeptide in treating digestive tract disorders.

Motirin is a substance that is isolated from pig's upper intestinal mucosa by Brown JC et al. And its amino acid binding sequence is determined and is a kind of peptide hormone (Gastroenterology 62, 401-404). (1972) and the Canadian Journal of Biochemistry (Can. J. Biochem.). 52, pp. 7-10 (1974). The pig's motirin consists of 22 amino acids and has a molecular weight of about 2,700.

On the other hand, with respect to the human-derived motirin, the inventors of the present invention confirmed that the cDNA clone was isolated and its structure was determined, and the amino acid binding sequence was found to be the same as that of pig-derived motorrin. (Japanese Patent Laid-Open No. 63-276489 ( A), corresponding US Patent Application No. 07/190849 and European Patent Application No. 88107108.81).

As the physiological action of motirin, it is well known that the action of hypertrophy of the digestive tract and the smooth muscle contraction of the digestive tract. Among these actions, for example, the action of shortening of the excretion time in the gastrointestinal tract has been reported (Gastroenterology Vol. 80, pp. 456-460 (1981)), and also the digestive tract smooth muscle. As for the contractile action, it is known that the gastric and duodenum of rabbits and humans exhibit strong contractile action without depending on the neural pathway. Motirin has been reported to be effective in treating or diagnosing gastrointestinal disorders after export, since no specific side effects have been reported. Note that prostagrandin is used for the treatment of intestinal palsy, although the drug has relatively strong side effects in this regard. The position of motorine is methionine, but it is called leucine or norleucine. Polypeptides having an amino acid structure modified by chemical synthesis have been reported to exhibit the same physiological activity as pure natural pig mortyrin (Scand. J. Gas troenterology, Vol. 11, pp. 119 to 203 (1976)). Therefore, the motirin residue at position 13 is thought to have little effect on the activity of the motirin.

According to the conventional technique, since mortyrin is generally derived from pigs and is obtained by extraction, mass production was extremely difficult.

On the other hand, even when the chemical method is used, since motirin is 22 polypeptides, it is difficult to obtain a large amount and low cost. In other words, although motirin is expected to be effective in the treatment of the digestive tract, its productivity is so poor that it has not been widely used in clinical applications.

Therefore, studies have been reported to apply so-called biotechnology to produce polypeptides having a motirin-type physiological activity at low cost (Japanese Patent Laid-Open Nos. 63-71195 (A) and 63-208006 (A)). In addition, there have been reports of developing pseudomotinins that exhibit higher physiological activity than motirin itself (Japanese Patent Laid-Open No. 61-26559 (B)).

In the above method using biotechnology, it has a step of cleaving using cyanogen bromide, and at this time, an operation of removing an excess peptide chain containing the generated C-terminal homoserine residue by enzymatic treatment is used. . However, such enzymatic treatment reduces the desired yield of pseudomotirin, increases production cost, and makes mass production of pseudomotirin difficult.

It is a main object of the present invention to provide new motirin-type polypeptides and salts thereof which have the same or higher physiological activity as motirin.

A first side object of the present invention is to provide a therapeutic agent for digestive tract containing an external drug, ie, a motirin-type polypeptide or a salt thereof as an active ingredient.

A second side object of the present invention is to provide a method for producing a motirin-type polypeptide and a salt thereof, which can be prepared more easily than conventional processes and commercialized at a reasonable price and in large quantities.

A third side object of the present invention is to provide a new recombinant DNA capable of performing the above process.

A fourth additional object of the present invention is to provide a plasmid expressing a motirin-type polypeptide by inserting the recombinant DNA.

The pharmacologically active moiety of motirin was found to span the region from the N-terminus to the 15-position of its amino acid binding sequence (peptide chemistry 1977, pp. 171-176 (1978)). The region is characterized by the presence of seven residues of the hydrophobic amino acids of phenylalanine (duplex), valine, proline, isoleucine, glycine and leucine in positions 1-10.

Under the assumption that hydrophobicity in the region has a relationship to the pharmacologic activity of motirin, that is, it binds to the motirin receptor on the cell membranes of the clonal smooth muscle of rabbits (Regulatory Peptides, Vol. 15, 146). P. 153 (1986)), the present inventors have synthesized various pseudomotilines, taking amino acids mainly from the 1-10 positions, and finally replacing the amino acid residues with the appropriate amino acid residues in the active motirin zone. It has been found that pharmacological activity can be increased by.

In addition, the inventors accidentally found that the polypeptide obtained by the cleavage process with cyanogen bromide and the other enzyme having the same or higher pharmacological activity than the original motirin was obtained by the non-enzymatic polypeptide itself. This means that enzymatic removal of amino acid residues, including homoserine (including homoserine lactones, hereinafter referred to as Hse), is not required.

Therefore, according to the present invention the above object is a general formula

(Wherein Z ₁ , Z ₂ and Z ₃ are amino acid residues having a hydrophobic side chain, A ₁ and A ₂ are amino acid residues that can take the transformation structure in the polypeptide chain, and B ₁ , B ₂ and B ₃ are aromatic rings) Amino acid residue with a side chain, C is Gln, Glu or Asp, D is Asn, Glu or Asp, X 'is an amino acid residue other than Met, E is Gln, Lys or Arg, Y is OH, homoserine lactone Or a peptide chain having an amino acid residue of 10 or less and having a homoserine or a homoserine lactone at the C-terminus) and salts thereof.

Examples of the amino acid having a hydrophobic side chain include valine, isoleucine, and leucine. Examples of amino acids that can have a modified structure in the polypeptide chain include proline, glycine, asparagine, and serine.

The reason X ', the amino acid residue at position 13, is an amino acid residue other than methionine is that X' is methionine, and when the fusion protein is treated with cyanogen bromide, cleavage occurs at the Met position at position 13, so It is because the desired polypeptide which has is not obtained. However, the amino acid residue at position 13 may also be Met, in which case the appropriate residue must be protected by a protecting group with a protecting group to prevent cleavage at that position due to cyanogen bromide treatment, and the protected Met at position 13 The resulting polypeptide with residues should be treated with cyanogen bromide to form a homoserine lactone residue at the C terminus, followed by removal of the Met protecting group.

Thus, the polypeptide according to the invention is of the general formula

및 Y는 전술한 바와 같은 의미이고, X는 선택적인 아미노산 잔기임)으로 표시되고, 모티린형 생리활성을 갖는다.(here,

And Y is as defined above and X is an optional amino acid residue.

A first, additional object of the present invention is obtained by a pharmaceutical composition for treating the digestive tract, containing an effective amount of at least one polypeptide represented by the general formula (I) and salts thereof in association with a medically acceptable carrier or excipient. Can lose.

Among the polypeptides represented by the general formula (I), a general formula

(Wherein C, X ', D and E are as mentioned above, A' is Pro, Gly, Asn or Ser, B 'is Gly, Asn or Ser, Y' is homoserine, homoserine, lactone , Or a peptide chain containing homoserine or homoserine lactone at the C-terminus and having at least 6 non-charged polar amino acid residues at its terminus DNA, amino acid binding sequence represented by the general formula (II), single-stranded DNA encoding methionine at the C terminus, and complementary single-stranded DNA, respectively, were synthesized, and the double-stranded DNA was prepared using the single-stranded DNA having a specific restriction enzyme recognition site. Respectively, add a predetermined restriction region of the expression vector using restriction enzymes, reconstruct the plasmid having the multiple bound genes, and transform the microorganisms using the reconstructed plasmid, A polypeptide having an amino acid binding sequence represented by the above formula (II) having no methionine at the C-terminus is produced as part of a fusion protein, and then treated with the same cyanogen bromide, followed by destruction of the microorganism, to give a general formula from the fusion protein. The second additional object of the present invention can be achieved by isolating the polypeptide of II) and then fractionating to separate the desired polypeptide of formula (II).

The reason why the Y terminus of the C terminus in the general formula (II) is a peptide chain having homoserine, homoserine lactone or amino acid residues of 10 or less is obtained by treating the fusion protein with cyanogen bromide to have the motirin type activity. This is because the desired polypeptide can be obtained. As non-polar amino acids, asparagine, glutamine, threonine, and serine are suitable for increasing the incidence of the desired polypeptide.

Tandem is used to prepare a polypeptide having an amino acid binding sequence of the general formula (II) having no methionine at the C terminus (Met is removed by treatment with cyanogen bromide at the C terminus) as part of the fusion protein. As a tandem arrangement, it is important to ligation a plurality of polypeptides having Met at the C terminus to express the motirin-type polypeptide as a polymerized protein. This can be done using the technique given in Japanese Patent Application No. 63-208006 (corresponding to US Patent Application No. 07/390149 and European Patent Application No. 89115199.5).

Each motirin-type polypeptide arranged in tandem has methionine at the C terminus, because the treatment due to the use of cyanogen bromide causes cleavage of the polymerized protein at each methionine site, thereby separating the polymerized protein into a structural monomer protein. Because it becomes.

As described above, a third additional object of the present invention is to formulate a leader array polypeptide having at least six or more non-charged polar amino acid residues, followed by the general formula (II) having no methionine at the C terminus. It can be achieved with recombinant DNA encoding a motif-type motirin-type polypeptide having an amino acid binding sequence of). In this case, it is preferable that a plurality of motirin-type polypeptides having Met at the C terminus of each polypeptide are arranged in tandem.

Finally, a fourth additional object of the present invention can be achieved with the expression plasmid into which the recombinant DNA is inserted.

Now, the present invention will be described in more detail in connection with Examples, Pharmacological Activity Test Examples and Formulation Examples.

In addition, below, the production of a motirin-type polypeptide in which methionine at the 13-position of the motirin is converted to leucine will be described. However, when the fermentation method is used, other motirin-type polypeptides can be prepared in the same form with amino acid residues other than methionine. To protect the methionine group by chemical synthesis method, to protect the cleavage at the position, and then treated with cyanogen bromide to form homoserine, homoserine lactone at the C-terminus, and then remove the protecting group for methionine group Note that you can.

Example 1

First, the polypeptide which has the following amino acid binding sequence was hip-hopped using the peptide synthesis | combination agent (Applied Biosystems company, type 430A).

Where R ₁ is

a) Met-Gly-Ser,

b) Asp-Gly-Ile-Phe-Met-Gly-Ser, or

c) Arg-Ile-Phe-Met-Gly-Ser.

Each synthesized polypeptide sample was purified by HPLC using a Waterbond Co. microbondasphere C-18 column (19 mn x 15 mn).

Eluent: linear gradient of 30% to 60% acetonitrile in 0.1% trifluoroacetic acid (30 minutes)

Flow rate: 7.0 ml / min

Each purified sample (10 mg) was weighed, dissolved in 30 ml of 70% formic acid solution, 50 mg of cyanogen bromide was added, and then reacted at 37 ° C. for 16 to 24 hours. Then, 200 ml of distilled water was added and lyophilized to remove formic acid and cyanogen bromide. Subsequently, the result was purified by HPLC using the Waters MicroBondasphere C-18 column under the same conditions as described above.

The main peak portion on the HPLC was recovered and lyophilized, and a portion thereof was treated with an Applied Biosystems peptide sequencer. Each sample was found to be correctly cleaved at the methionine position, and the R _{1 at the} C terminus was modified. The polypeptide was as follows.

A) Hse,

B) Asp-Gly-Ile-Phe-Hse, or

C) Arg-Ile-Phe-Hse.

In this example, a method for producing a motirin-type polypeptide is described. Here, the methionine at position 13 of the motiline is modified with leucine, but other motiline-type polypeptides having an amino acid residue other than methionine at position 13 are conventional. It can be manufactured by fermentation method. It should also be noted that motirin-type polypeptides in which some of the amino acid residues are modified can also be obtained by synthesis in the same manner as described above, such as peptide synthesis, cleavage with cyanogen bromide and purification by HPLC.

Example 2

(1) Synthesis of DNA encoding a motirin-type bioactive polypeptide.

A motirin-type polypeptide substituted with methionine Leu at 13-position and Hse-added at 23-position has the following amino acid binding sequence.

When a fusion protein produced by a microorganism is treated with cyanogen bromide, it is known on the polypeptide that the Hse residue at position 23 is formed by modification of the Met residue.

Therefore, a double-stranded DNA fragment represented by the following formula (1) was synthesized, which includes a nucleotide encoding an amino acid binding sequence having a Met residue at position 23 of the amino acid binding sequence, and the plurality of leading polypeptides via Met. Has a nucleotide sequence encoded by the non-charged polar amino acid magnetic (Met-Thr-Met-Ile-Thr-Asn-Ser-Gln-Gln-Gln-Gln-Gln-Gln-Ile-Phe-Met) at the N terminus, The C-terminal has a nucleotide sequence encoding a polypeptide having an amino acid binding sequence of Gly-Ile-Leu.

Formula (1)

The double-stranded DNA represented by the above formula (1) is synthesized by the following operation. First, six single-stranded DNAs each having the following amino acid binding sequences were synthesized using a DNA synthesizer (Gene Assembler, Pharmacy AB, Sweden).

Of these, it should be noted that each DNA pair of the formulas (2) and (7), (3) and (6), and (4) and (5) is complementary DNA except for a part thereof.

Each single-stranded DNA represented by the formulas (2) to (7) was treated with polynucleotide-kinase, respectively, to phosphorylate the 5'-end, and then the formulas (2), (7), (3) and (6). ) And each of the complementary DNA pairs of (4) and (5) were annealed, respectively, and conjugated by tandem with the formed triplet DNA ligase to obtain a DNA fragment represented by the formula (1).

(2) Assembly of DNA fragments into expression vectors.

An operation of assembling the synthesized DNA fragment obtained in the above (1) into an expression vector (plasmid) will be described with reference to FIG.

The plasmid was cleaved by reacting the restriction enzymes HindIII and EcoR I to a plasmid (pTM1) having a Trp promoter used for expression in E. coli.

In the obtained cleavage site, the synthetic DNA fragment obtained in the above (1) was ligated using DNA ligase to reconstruct the recombinant plasmid. This reconstituted plasmid was named pTMH1. The protein synthesis was designed to begin at the position 10 residues from the SD sequence downstream of the Trp promoter. Therefore, when introduced into E. coli or the like, the production of protein can be achieved by the use of indole acrylic acid (IAA) or the like. The reconstituted vector will also have a recognition site for restriction enzymes BglII and Bam H I.

(3) Construction of a recombinant plasmid having two or more motirin-type genes.

See FIG. 2 for this.

First, the recombinant plasmid (pTMH1) obtained in the above (3) was treated with the restriction elements EcoRI and BamHI to recover the fragments by cleaving the motirin-type gene at the upstream site and at the 1 site in the vector. Another plasmid (pTMH1) was then treated with the restriction enzymes EcoRI and Bgl2 to cleave the same site as the site in the vector and the downstream site of the motirin-type gene portion to be common with the site of the upstream site described above. The cleaved fragment was ligated with DNA ligase at this cleavage site, thereby obtaining expression plasmids of two motirin-type genes, both of which were linked through a peptide of Gly-Ile-Phe-Met. The resulting recombinant plasmid was called pTMH2.

In this case, the cleavage site by restriction enzymes BamH I and BgIII was ligated with DNA ligase, but the ligated site became a site that could not be recognized by both enzymes. Thus, by repeating the above operation, a reconstituted plasmid having the leader peptide sequence upstream of the motirin-type gene was linked in random number and through methionine upstream.

(4) Production of Motirin-type Polypeptide-Containing Fusion Proteins

A reconstituted plasmid (pTMH4, prepared in the same manner as described in (3) above) containing four linked motirin-type genes in tandem was placed in E. coli (HB101) and transformed (this transformant is called E. coli MH4). And the International Depository RFI of Japan under the International No. FERM BP-2555). The transformants were aerated at 30 ° C. in a medium containing 50 μg / m1 of ampicillin (for example, a medium prepared by adding 0.5% casamino acid to M9 medium). When the A ₅₅₀ of a 0.6-0.8, the addition of indole acrylic acid (IAA), and made the final concentration of 15μg / ml.

The cells were then incubated for 16 hours, followed by centrifugation (8,000 rpm, 4 ° C., 5 minutes) to recover the cells. A portion of the cells were harvested and analyzed by 15% polyamide gel electrophoresis. Proteins accounted for about 30% of the total protein. This is presumably because the lead peptide sequence in the synthetic DNA assembled into the plasmid promotes granulocyte formation with high efficiency and is very stable to protease.

(5) Isolation and Purification of Motirin-type Polypeptides

Paste type cells (about 30 ml) obtained in the above (4) were suspended in 200 ml of cold acetone, filtered through a glass filter, the filtrate was suspended in 300 ml of saline solution, and then placed in a high pressure homogenizer or sonicated. Cells were crushed. The residue of the cells was centrifuged for 15 minutes at 10,000 rpm.

This pellet contains a fusion protein of a leader array peptide and a motirin-type polypeptide tetramer. Dissolve the pellets (about 500 mg of protein) in 30 ml of 70% formic acid. 500 mg of cyanogen bromide was added to this solution and reacted at 30 ° C. overnight. Then, 40 g of NaOH and 200 ml of distilled water were added to adjust the pH of the solution to three. The solution was centrifuged (1,000 rpm, 20 minutes) to recover the aqueous layer.

The solution was desalted with a Sephadex G-15 column to remove formic acid and cyanogen bromide, and the cation exchange chromatography of the motirin-type polypeptide fragment was followed by a micro bonder C-18 column (19 mm x 15 cm). Purification was carried out by HPLC under the following conditions.

Eluent: linear gradient of 30-60% acetonitrile in 0.1% trifluoro acetate for 30 minutes.

Flow rate: 7.0 ml / min.

The main peak portion in HPLC was recovered, lyophilized, a portion of the dried powder was collected and confirmed using a peptide sequencer of Applied Biosystems. The sample had Hse at the C terminus and had the correct amino acid binding sequence. It was confirmed that it is a desired motyrin type polypeptide.

By the same procedure as described above, 400-500 mg of the desired motirin-type polypeptide was obtained by culturing the reconstituted Escherichia coli (E. coli MH4).

Biological activity test example 1

Each of the motirin-type polypeptides according to the present invention (Example 1, wherein R ₁ is Hse, Asp-Gly-Ile-Phe-Hse or Arg-Ile-Phe-Hse) is used as a test substance, and a conventional pure [leucine ¹³ Intestinal contractile activity was measured according to the Magnus method (J. Pharm. Pharmac., Vol. 28, pp. 650-651 (1976)) using the upper intestine of rabbits as a control. . The contraction with ^10-6 M acetylcholine was set at 100% and the activity of the test and control was compared.

The results are shown in FIG. 3, and as shown in the figure, the intestinal contractile activity exhibited by each polypeptide obtained by the present invention was found to be equal to or higher than that of [leucine ¹³ ] -motirin.

Biological activity test example 2

Intestinal contractile activity of various pseudomotirins prepared in the same manner as in Example 1 was measured by the Magnus method used in the physiological activity test example 1, and each ED ₅₀ was determined assuming 100% pure motirin shrinkage. And compared to that of pure motirin.

The results are shown in the first table. All of the pseudomotirins given in the table are indicated as Hse at the C terminus, but it was noted that the pseudomotirins have substantially the same level of contractile activity when Hse is homoserine lactone or their sodium salt. In addition, it exhibited the same contractile activity as having the Hse residue at the C-terminal pseudomotirin group converted to the OH group.

From the results shown in Table 1, it can be seen that even if the pharmacological activity of human motirin depends on the amino acid binding sequence near the N terminus, the activity can be increased by changing one or more amino acid residues. The relationship between the amino acid binding sequence activity of can be said as follows.

A) The pseudomotirin substituted with Gly, Ser or Asn residues that can take a modified structure in the polypeptide chain of Pro and 8-position Gly, which is the 3rd position of the original motif, has the same or more morphology of the original Although exhibiting high activity, the activity is significantly reduced when the amino acid residue at the position is replaced with Ile, which is difficult to take a meander structure.

In this case, the pseudomotirin in which Pro, which is the 3-position of the original motirin, is substituted with Gly, shows quite high activity, that is, about twice as much as the original motirin.

B) Although the original positions of Motirin, Val 2, Ile 4 and Leu 10 are amino acids with hydrophobic side chains, the pseudomotirins having the interchanged structure of these amino acids are the same as or more than those of the original High activity.

Although the Phe and 1.5 positions of the original motirin are amino acids with side chains having aromatic rings, the pseudomotirins having the interchanged structure of these amino acids show the same activities as those of the original motirin. When the amino acid at this position is exchanged for another amino acid, the activity is markedly reduced.

D) The C terminus of the original motirin is believed to consist of charged amino acid residues or polar amino acid residues, and it is important to maintain activity in vivo, although not particularly the main active. Therefore, when the motirin having an amino acid structure that increases the positive or negative charge at the C terminus was examined, almost no change in activity was found. This suggests that if the charge is balanced, some degree of amino acid exchange near the C terminus does not significantly affect activity.

Biological activity test example 3

[Leucine [Leucine obtained by chemical method using] -motirin-homoserine (Example 1, R: Hse) as a test substance ] -Motirin was used as a control, and adjusted to an amount of 20 μg / kg / hour, and bowel contraction activity (upper intestine, plant and ileum) was measured by the Balloon method using an unconscious dog. The results are shown in FIGS. 4A and 4B. As can be seen from the drawings, the polypeptide ([leucine] ] -Motirin-homoserine) is the reference compound [leucine It was confirmed that the contractile activity is significantly higher than that of] -motirin, and that the contractile activity of the polypeptide of the present invention is expressed in a natural manner.

In addition, the polypeptide having a homoserine lactone residue at position 23 of the present invention has the same or similar physiological activity as having a homoserine residue at position 23.

In addition, a polypeptide having less than 10 amino acid residues of the present invention and containing an Hse residue at the C terminus exhibits the same or the same biological activity as having a homoserine residue at position 23.

Biological activity test example 4

[Leucine 13] -Motirin- Homoserine (Example 1, R: Hse, Test Example 1) and its sodium salt (Test Example 2) as a test substance, [Lucine ] -Motirin was used as a control material, and adjusted to an amount of 2.0 μg / kg / hour, and the intestinal contraction activity of the upper small intestine was measured by a balun method using an unconscious dog used in physiological activity test example 3 to determine ED. Next, control activity was assumed to be 100%. The results are shown in the second table below. As can be seen from this table, it can be seen that the activity of the test substance is 10-20% higher than the control substance.

Biological activity test example 5

[Leucine ] -Motirin-homosehin (Example 1, R: Hse) and its sodium salt as test substance, [leucine ] -Shrinkage of the small intestine by the balun method using the unconscious dog used in physiological activity test example 3 using motirin as a control and an externally accepted prostaglandin F for treating the digestive tract of Ileus et al. Activity was measured, ED was measured, and then the prostaglandin activity was compared at assuming 100%. The results are shown in FIG. As can be seen from the figure, the polypeptides and salts thereof according to the invention show activity at significantly lower concentrations compared to the widely acceptable formulations of prostaglandins, resulting in significantly higher doses when the same effect is expected. Can be reduced.

In Figures 4A and 4B, polypeptides according to the invention, in particular [leucine Considering that] -motirin-homoserine exhibits intestinal contractile activity, it can be seen that the material of the present invention is superior to the conventional prostaglandin preparations in terms of its stability and functionality.

Formulation example

Polypeptides according to the present invention in purified water ([Leucine ] -Motirin-homoserine, Example 1, R: Hse) solution was aseptically dispensed into vials to contain lmg of polypeptide in each vial. After lyophilization, the vial was sealed to obtain a dry powder. When used, the powder was dissolved in physiological saline for injection.

Claims (4)

Formula (I)

[Wherein Z ₁ means Val or Leu, Z ₂ means Ile or Leu, Z ₃ means Leu or Ile, A ₁ means Pro or Gly and A ₂ means Gly or Ser And B ₁ , B ₂ and B ₃ mean Phe or Tyr, C means Gl, Glu or Asp, X means Leu, D means Asn, Glu or Asp, and E means Gln , Lys or Arg, and Hse means homoserine or homoserine lactone. Formula (I)

[Wherein Z ₁ means Val or Leu, Z ₂ means Ile or Leu, Z ₃ means Leu or Ile, A ₁ means Pro or Gly, and A ₂ means Gly or Ser B ₁ , B ₂ and B ₃ mean Phe or Tyr, C means Gl, Glu or Asp, X means Leu, D means Asn, Glu or Asp, and E is Gln, Lys or Arg, and Hse means homoserine or homoserine lactone]. Formula (Ⅱ)

[Wherein A means Pro, Gly, Asn or Ser, B means Gly, Pro, Asn or Ser, C means Gln, Glu or Asp, X means amino acid residues other than Met and , D means Asn, Glu or Asp, E means Gln, Lys or Arg, and means Hse homoserine or homoserine lactone.

Single-stranded DNA encoding the leader array polypeptide of the amino acid sequence represented by

Single-stranded DNA encoding a motirin-type polypeptide having an amino acid sequence represented by [wherein A, B, C, X, D and E are as described above], and a base sequence complementary to the base sequence in each of these terminal DNAs Single monoclonal DNAs were synthesized, single monoclonal DNAs were prepared, and two kinds of restriction enzymes were cut on a plasmid having an E. coli Trp promoter, and the above-described synthetic double-stranded DNA was conjugated to the cleavage point to form a plasmid. The plasmid for expression thus obtained was transformed by inserting microorganisms and transformed at the same time, followed by culturing, and expressed by the formula (II), except that the C-terminal polypeptide having an amino acid sequence of Met but not Hse was fused protein. Individual polypeptides represented by the above formula (II) by producing them as part of and subsequently destroying the fusion protein producing microorganisms and treating with cyanogen bromide Motif rinyong method of producing a polypeptide isolated from the fusion protein and the de characterized in that the purified fraction treatment. The DNA fragment encoding the leader array polypeptide moiety and the motirin-type polypeptide moiety is recovered by treating the reconstructed plasmid as two kinds of restriction enzymes again, while cleaving by acting two restriction enzymes on plasmid pTM1. By conjugating the recovered DNA fragment to the cleavage point, the plasmid is conjugated by two tandems and via a spacer peptide of amino acid sequence of Gly-Ile-Phe-Met. The plasmid is cut again, the above-mentioned introduction procedure of the motif-type polypeptide is repeated, and then inserted into the microorganism.

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