JPH0722517B2 - Method for producing motilin-like polypeptide, recombinant DNA therefor and expression plasmid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a motilin-like polypeptide, particularly a motilin-like polypeptide having Hse at the C-terminus, and a recombinant DNA and an expression plasmid therefor.

The polypeptides according to the invention can be used as medicaments, in particular for the treatment of gastrointestinal disorders.

(Prior Art) Motilin is a substance whose structure has been determined and isolated for the first time from the upper small intestinal mucosa of pigs by Brown et al. And is a kind of peptide hormone ["Gastroenterology" Vol. 62, pp. 401-404 ( 1972) and "Can. J. Biochem." Vol. 52, pp. 7-10 (1974)]. This porcine motilin is composed of 22 amino acids and has a molecular weight of about 2700.

On the other hand, regarding human-derived motilin, the present inventors isolated the cDNA clone and determined the structure, and as a result, it was clarified that its amino acid sequence was identical to that derived from pig ( Wish sho 62-10975
7).

As the physiological actions of motilin, gastrointestinal motility enhancement action and digestive tract smooth muscle contraction action are well known. Among these effects, as for the gastrointestinal motility enhancing effect, for example, an effect of shortening gastric emptying time has been reported [[Gast
roeeterology ", Vol. 80, pp. 456-460 (1981)], and as for the contractile action of gastrointestinal smooth muscle, it can bring about strong contraction to the antrum of the stomach and duodenum of rabbits and humans without depending on the neural pathway. Therefore, since motilin enhances gastrointestinal motility and no particular side effects have been reported, it has been considered that motilin is effective for treatment of gastrointestinal disorders and diagnosis of gastrointestinal disorders after surgery ( Incidentally, prostaglandins and the like are currently used for the treatment of postoperative intestinal paralysis, etc., but there is a problem in that side effects are strong).

Although 13th position in the amino acid sequence of motilin is methionine, it has been reported that a chemically synthesized polypeptide having an amino acid structure in which methionine is converted to leucine or norleucine also shows physiological activity similar to that of motilin, [[Sc
and.J. Gastroenterology "Vol. 11, pp. 119-203 (1976
, Etc.], and methionine at position 13 is considered to have little effect on the activity.

Furthermore, it has been clarified by the present inventors that a motilin-like polypeptide containing Hse at the C-terminal exhibits physiological activity equivalent to or higher than motilin (Japanese Patent Application No. 64-286).

(Problems to be Solved by the Invention and Objects of the Invention) According to the conventional technique, motilin is generally derived from pigs and is obtained by extraction, and therefore, mass production was extremely difficult. On the other hand, even when the chemical synthesis method is used, motilin is a polypeptide having 22 amino acids, and thus it is difficult to obtain motilin in large quantities and at low cost. That is, although motilin is expected to be effective in treating gastrointestinal disorders, its productivity has been a bottleneck and has not been widely used for clinical treatment.

Therefore, studies have been conducted repeatedly to inexpensively produce a polypeptide having a motilin-like activity by applying so-called "biotechnology" (Japanese Patent Application Laid-Open No. 63-71195 and the patent application developed by the present inventors). 63-208006, etc.).

However, the above-mentioned methods utilizing biotechnology have a step of cleaving with bromocyan, and an operation of removing the peptide containing Cse Hse generated at this time by enzymatic treatment is carried out. The enzyme treatment process causes a decrease in yield and an increase in cost, and thus has been a problem in actual mass production. Therefore, the inventors of the present invention have conducted diligent research and as a result, as described above (Japanese Patent Application No. 64-28).
6 specification), a motilin-like polypeptide having Hse at the C-terminus has physiological activity equal to or higher than that of motilin, and thus it has been recognized that the removal operation of the Hse portion is unnecessary. .

The present invention has been made in view of these recently developed methods, and the essential purpose thereof is to provide a method for more easily producing a motilin-like polypeptide.

An additional but important object of the present invention is to provide a method for producing motilin-like polypeptides in a highly efficient and inexpensive manner.

Another object of the present invention is to provide a novel recombinant DNA used for carrying out the above production method.

Still another object of the present invention is to provide an expression plasmid incorporating the above recombinant DNA.

(Means and Actions for Solving Problem and Achieving Object) According to the present invention, the above-mentioned problem is solved by the formula (I) Phe-Val-A-Ile-Phe-Thr-Tyr-B-Glu-Leu-C.
-Arg-X-Gln-Glu-Lys-Glu-Arg-D-Lys-Gly-
E-Hse (wherein A means Pro, Gly, Asn or Ser, B means Gly, P
ro means Asn or Ser, C means Gln, Glu or Asp, X means an amino acid residue other than Met, and D means Asn or Gl.
u or Asp, E means Gln, Lys or Arg, H
se means homoserine or homoserine lactone residue), and is a method for producing a motilin-like polypeptide represented by the formula: Met-Thr-Met-Ile-Thr-Asn-Ser-Gln-Gln-Gln-
Single-stranded DNA encoding a leader polypeptide having an amino acid sequence represented by Gln-Gln-Gln-Ile-Phe-Met, and Phe-Val-A-Ile-Phe-Thr-Tyr-B-Glu-Leu- C
-Arg-X-Gln-Glu-Lys-Glu-Arg-D-Lys-Gly-
E-Met (wherein A, B, C, X, D and E have the above-mentioned meanings), a single-chain DAN encoding a motilin-like polypeptide having an amino acid sequence, and each of these single-chains Each of the single-stranded DNAs having a nucleotide sequence complementary to the nucleotide sequence in the DNA was synthesized, and Japanese-stranded DNA was prepared using these single-stranded DNAs, while two types of restriction enzymes were added to the plasmid having the Escherichia coli Trp promoter To cleave it, and join the above-mentioned synthetic double-stranded DNA at the breakpoint to reconstruct a plasmid, and the expression plasmid thus obtained is introduced into a microorganism and transformed and cultured. A polypeptide having the amino acid sequence represented by the above formula (I) but having Met instead of Hse at the C-terminus is produced as a part of the fusion protein, and thereafter the fusion protein-producing microorganism is destroyed and treated with bromocyan. What to do Is solved by a method for producing a motilin-like polypeptide, characterized in that the individual polypeptide represented by the above formula (I) is separated from the fusion protein, fractionated and purified. The purpose is achieved.

Here, a polypeptide having the amino acid sequence represented by the above formula (1) and having a methionine (Met) at the C-terminus (converted to Hse after cleavage with bromocyan) is used as a fusion protein in a microorganism. In order to produce a large amount of the motilin-like polypeptide, a plurality of motilin-like polypeptides having a C-terminal methionionine (Met) gene are joined in tandem, whereby the motilin-like polypeptide is expressed as a polymer protein. Is important, connect multiple motilin-like polypeptide genes in tandem,
The incorporation itself into an expression vector has already been reported by the present inventors (Japanese Patent Application No. 63-208006).
Specification), this method can be utilized. The amino acid sequence between the motilin-like polypeptide moieties may be Met at the C-terminus. This is because a polymer protein consisting of a motilin-like polypeptide bound via Met is cleaved at the Met portion and separated into monomers when treated with bromocyan.

In the formula (1) showing the motilin-like polypeptide produced by the method of the present invention, X which is the amino acid residue at position 13 is
This site is considered to be an amino acid residue other than Met.
If et is the latter step, i.e., when the produced fusion protein is treated with bromocyan, the desired polypeptide having motilin-like activity cannot be obtained because cleavage also occurs at the Met position 13. is there. In the present invention, the last amino acid in the leader sequence peptide is composed of methionine (Met) because the leader sequence portion can be easily cleaved in a later step, that is, by treatment with bromocyan. Is. A single-chain D encoding a polypeptide having a leader sequence portion and an amino acid sequence represented by the formula (1) and having a C-terminal Met (converted to Hse after being cleaved by Bromocyan treatment)
NA can be divided and synthesized, which facilitates the synthesis.

The recombinant DNA according to the present invention is represented by the above-mentioned formula (I) at the C-terminal side of the above-mentioned leader sequence polypeptide, provided that C
It is characterized in that a motilin-like polypeptide whose end is Met rather than Hse is linked. In this case, it is advantageous that a plurality of motilin-like polypeptides, for example four, are linked after the leader sequence polypeptide,
In this case, it is important that the spacer peptide that connects the motilin-like polypeptides to each other is not too long and that it has a Met at the C-terminal to facilitate its separation. For example, Gly-Ile- Peptides encoding the amino acid sequence Phe-Met are preferred.

The leader sequence polypeptide in the recombinant DNA according to the present invention is rich in the uncharged polar amino acids asparagine (Asn), glutamine (Gln), threonine (Thr) and serine (Ser). Recombinant DNA with peptide placed in front of motilin-like polypeptide
Is incorporated into an expression plasmid, incorporated into a host cell, and this transformed cell is cultured, the above leader sequence remarkably promotes the formation of granules, and the production amount of the desired gene protein increases.

On the other hand, the expression plasmid according to the present invention is characterized in that the recombinant DNA as described above is incorporated therein.

(Examples, etc.) Next, the present invention will be described in more detail with reference to production examples and pharmacological activity test examples. In the following, the production of a motilin-like polypeptide (L-leucine-13-motilin-homoserine) in which the 13th position of motilin is converted to leucine (Leu) and Hse is added to the 23rd position will be described, but the C-terminal But
Note that with Hse, other motilin-like polypeptides (without methionine as an amino acid residue) can be similarly prepared.

Production Example 1 (1) Synthesis of DNA encoding a motilin-like physiologically active polypeptide A motilin-like polypeptide in which the 13th position is converted to Leu and Hse is added to the 23rd position has an amino acid sequence as shown below. ing.

In the above-mentioned polypeptide, Hse at position 23 is converted from Met by treatment with bromocyan after the above-mentioned polypeptide is produced by the microorganism.

It is known.

Therefore, a leader peptide (Met-Thr) containing a nucleotide sequence encoding an amino acid sequence in which the 23rd position in the above-mentioned amino acid sequence is Met and having a large number of uncharged polar amino acid residues through methionine at its N-terminal side is included. -Met-Ile-
Thr-Asn-Ser-Gln-Gln-Gln-Gln-Gln-Gln-Ile-
A double-stranded chain represented by the following formula (2) having a base sequence encoding Phe-Met) and a base sequence encoding a peptide consisting of an amino acid sequence (Gly-Ile-Leu) on the C-terminal side: A DNA fragment was produced.

The double-stranded DNA represented by the above formula (2) was synthesized as follows.

First, using a DNA synthesizer "Gene Assembler" manufactured by Pharmacia, six types of single-stranded DNA having the following base sequences were synthesized. Of these, equations (3) and (8),
(4) and (7) and (5) and (6) are DNAs complementary to each other except for a part of the ends.

Formula (3) 5'-AATTCATGACCATGATTACGAACTCACAACAACAACAACA Formula (4) 5'-GATCTTCATGTTCGTTCCGATCTTCACCTACGGCGAACTGCAG Formula (5) 5'-CGTCTGCAAGAAAAAGAGCGCAACAAAGGCCAGATGGGGATCCT
GTGATA formula (6) 5'-AGCTTATCACAGGATCCCCATCTGGCCTTTGTTGCGCTCTTTTT
CTTG formula (7) 5'-CAGACGCTGCAGTTCGCCGTAGGTGAAGATCGGAACGAACATGA
A formula (8) 5'-GATCTGTTGTTGTTGTTGTTGTGAGTTCGTAATCATGGTCATG The single-stranded DNAs represented by formulas (3)-(8) are each treated with a polynucleotide kinase to phosphorylate the 5'end, and then a complementary DNA pair is used. Some formulas (3) and (8),
(4) and (7) and (5) and (6) are each annealed to form three double-stranded DNAs, which are tandemly joined by DNA ligase to form a DN represented by the formula (2).
A fragment was obtained.

(2) Incorporation into expression vector The procedure for incorporating the synthetic DNA fragment obtained in the above item (1) into an expression vector will be described with reference to FIG.

The plasmid pTM1 containing the Escherichia coli Trp promoter is cleaved by the action of restriction enzymes Hind III and EcoR I, and the synthetic DNA fragment obtained in the above item (1) has the Trp promoter at the breakpoint by DNA ligase. The plasmid was reconstructed by conjugating with a plasmid for expression in E. coli. This reconstructed plasmid is named pTMH1 and is designed so that protein synthesis is initiated at a position 10 residues away from the SD sequence in the downstream region of the Trp promoter. Therefore, when this expression plasmid is introduced into Escherichia coli or the like, it is possible to induce the protein with indole acrylic acid (IAA) or the like.

In addition, the above reconstructed vector was constructed with restriction enzymes BglII and BamHI.
It is designed to have the recognition site of.

(3) Construction of expression plasmid containing two or more motilin-like polypeptide genes See FIG. 2 for this. First, the plasmid (pTMH1) obtained in the above item (2) was digested with the restriction enzyme EcoRI.
And BamHI and digested at the upstream region of the motilin-like polypeptide gene portion and at one site in the vector to recover the fragment. Next, the plasmid (pTMH1), which is the plasmid obtained in the above (2), is treated with restriction enzymes EcoR I and Bgl II separately from the digested plasmid to obtain the same plasmid as the above-mentioned site in the vector. The site and the downstream region of the motilin-like polypeptide gene part are cut so that they are common to the above-mentioned upstream region breakpoints, and the above-recovered fragment is ligated to the breakpoints with DNA ligase to reconstruct the plasmid. Then, an expression plasmid containing two motilin-like polypeptide genes (pTMH2)
Is obtained. In this case, the motilin-like polypeptide genes are linked to each other by a Gly-Ile-Phe-Met peptide.

In the above case, the breakpoint by the restriction enzyme BamH I and the breakpoint by the restriction enzyme Ba1 II are joined by DNA ligase, but this joining site is no longer the cleavage recognition site for both enzymes. Therefore, by repeating the above-mentioned cleavage and conjugation operations, an arbitrary number of motilin-like polypeptide genes are bound in tandem, and a plasmid having a leader peptide sequence upstream of methionine can be obtained.

(4) Production of Motilin-Like Polypeptide-Containing Fusion Protein A plasmid (pTMH4) containing the motilin-like polypeptide gene, which was prepared as described in the above item (3) and has four tandem linkages. ) E. coli (HB101)
To obtain a quasi-converted bacterium [this was deposited internationally at the Institute of Microbial Technology, Institute of Industrial Technology (Mikken Kenki No. 2555).
issue)]. This stromal-converting bacterium was subjected to aeration culture at 30 ° C. with a culture solution containing 50 μg / ml of ampicillin (for example, M9 medium supplemented with 0.5% casamino acid) and using a D type fermenter manufactured by Able. Indole acrylic acid (IAA) was added when A ₅₅₀ reached 0.6-0.8 to reach a final concentration of 15 μg / ml.

Then, the culture was continued for 16 hours and then centrifuged (8000rp
The cells were collected at m, 4 ° C. for 5 minutes). SDS polyacrylamide gel electrophoresis (1
5%), the desired fusion protein amounted to about 30% of the total protein. It is presumed that this is because the leader peptide sequence part in the synthetic DNA incorporated in the plasmid promotes the formation of granulocytes extremely efficiently and is hardly decomposed by protease.

(5) Separation and purification of motilin-like polypeptide The paste-like bacterial cells (corresponding to 30 ml) obtained in the above (4) are suspended in 200 ml of cold acetone and then filtered through a glass filter to remove the residue. The cells were suspended in 300 ml of physiological saline and subjected to high pressure homogenization or ultrasonic treatment to destroy the cells. The cell residue was pelletized by centrifugation at 10,000 rpm for 15 minutes. This pellet contains a fusion protein of a leader peptide and a motilin-like polypeptide tetramer.

Therefore, this pellet (protein amount about 500 mg) was dissolved in 30 ml of 70% formic acid solution, 500 mg of bromocyan was added to this solution, and the mixture was reacted overnight at a temperature of 30 ° C. After that, 40 g of NaOH and 200 ml of distilled water were added to adjust the pH to 3 or more, and then the supernatant was recovered by centrifugation (1000 rpmx20 minutes) and desalted using Sephadex G-15 to remove formic acid and bromocyan. After removal, the motilin-like polypeptide elution fraction was treated with cation exchanger chromatography.
Next, Water Bond C of Micro Bonder Sphere
Purification was carried out by HPLC using a -18 column (19 mm x 15 cm) under the following conditions.

Eluent: linear gradient of 30% to 60% acetoanitrile in 0.1% trifluoroacetic acid, 30 minutes) Flow rate: 7.0 ml / min Collect the main peak part by HPLC and lyophilize,
When a part of the sample was collected and examined by a peptide sequencer manufactured by Applied Biosystems, it was confirmed to be a motilin-like polypeptide having a correct sequence having Hse at the end.

By performing the above operation, 400-500 mg of the desired motilin-like polypeptide could be obtained by culturing 1 liter of E. coli.

Example of physiological activity test (measurement of intestinal contractile activity) Motilin-like polypeptide (L-
Leucine-13-motilin-homoserine) as a test substance, pure motilin obtained by the synthetic method as a reference substance,
Magnus method using rabbit duodenum [“J.Pharm.Pharma
c. "Vol. 28, pp. 650-651 (1976)", the intestinal contractile activity was measured and the results are shown in Fig. 3.

As is clear from this figure, acetylcholine (10 ^-6 M)
It was confirmed that the intestinal contractile activity of L-leucine-13-motilin-homoserine obtained by the present invention is about the same level as that of pure motilin, when the contraction by the method is 100%.

(Effect of the Invention) According to the present invention, a DNA fragment containing a nucleotide sequence encoding an amino acid sequence of a motilin-like gene is divided, chemically synthesized, and then bound to form a desired DNA fragment. , The synthesis of this DNA fragment is relatively easy. In this DNA fragment, a leader peptide sequence is present at the N-terminal side of the amino acid sequence encoding a motilin-like gene via methionine, and this motilin-like gene-containing DNA fragment is incorporated into a plasmid as a vector and the plasmid is incorporated into a microorganism. When incorporated and cultured, the above leader peptide promotes the production of the motilin-like active substance, so that the production efficiency is increased.

In addition, after the above leader peptide sequence, a plurality of motilin-like genes can be linked in tandem via Met, and thus a DNA containing a plurality of motilin-like genes can be incorporated into a plasmid and If the plasmid is taken up by a microorganism and cultured, naturally, the expression rate of the motilin-like active substance can be remarkably improved.

Moreover, according to the method of the present invention, since the purification treatment is only the treatment with bromocyan, it is extremely simple.

Therefore, the present invention enables inexpensive and large-scale production of a motilin-like physiologically active substance, and replaces prostaglandins etc., which are currently used for postoperative intestinal paralysis of patients and have strong side effects. It should provide safe medicines.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a construction mode of a gene recombinant plasmid having one motilin-like gene, which constitutes a main part of the method of the present invention, and FIG. 2 is similar to FIG. 1, except that the motilin-like gene is FIG. 3 is an explanatory view showing a construction state of a gene recombinant plasmid having two tandems, and FIG. 3 is an intestinal tract of L-leucine-13-motilin-homoserine produced by the method of the present invention and pure motilin obtained by a conventional method. It is a graph which shows the result of having measured contractile activity by the Magnus method.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kenichi Tanaka 35 Higashi-Tobori-cho, Higashi-ku, Nagoya-shi, Aichi Stock Company Sanwa Chemical Research Institute (72) Inventor Katsuya Fujimura 35 Higashi-Tobori-cho, Higashi-ku, Nagoya, Aichi Stock Company Sanwa Chemical Research Institute (72) Inventor Yuji Hayashi 35, Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi Stock Company Sanwa Chemical Research Institute (72) Inventor Yohei Kobayashi 35 Higashi-Tobori-cho, Higashi-ku, Aichi Prefecture Sanwa Co., Ltd. Inside the Institute for Chemical Research (72) Inventor Kiichi Sawai 35, Higashi-Tobori-cho, Higashi-ku, Nagoya City, Aichi Prefecture Within Sanwa Institute of Chemical Research (56) References JP 63-71195 (JP, A) JP 61-5788 ( JP, A) JP 61-173780 (JP, A) Biotechnology Letters, vol. 10, No. 11, P.I. 763-768 (1988)

Claims (6)

[Claims] 1. A formula (I) Phe-Val-A-Ile-Phe-Thr-Tyr-B-Glu-Leu-C.
-Arg-X-Gln-Lys-Glu-Arg-D-Lys-Gly-E-H
se (where A means Pro, Gly, Asn or Ser, B means Gly, P
ro means Asn or Ser, C means Gln, Glu or Asp, X means an amino acid residue other than Met, and D means Asn or Gl.
u or Asp, E means Gln, Lyn or Arg, Hse
Means a homoserine or a homoserine lactone residue), and is a method for producing a motilin-like polypeptide represented by: Met-Thr-Met-Ile-Thr-Asn-Ser-Gln-Gln-Gln-
Single-stranded DNA encoding a leader sequence polypeptide of the amino acid sequence represented by Gln-Gln-Gln-Ile-Phe-Met, and Phe-Val-A-Ile-Phe-Thr-Tyr-B-Glu-Leu -C
-Arg-X-Gln-Gln-Lys-Glu-Arg-D-Lys-Gly-
E-Met (wherein A, B, C, X, D and E have the above-mentioned meanings), a single-stranded DNA encoding a motilin-like polypeptide having an amino acid sequence, and each of these single-stranded DNAs. Synthesized single-stranded DNAs each having a nucleotide sequence complementary to the nucleotide sequence in the DNA, and used these single-stranded DNAs to prepare Japanese-stranded DNAs. On the other hand, two types of restriction were made on the plasmid containing the Escherichia coli Trp promoter. Cleavage is performed by the action of an enzyme, the synthetic double-stranded DNA is joined to the breakpoint to reconstruct the plasmid, and the expression plasmid thus obtained is transformed with the microorganism incorporated therein and cultured. The polypeptide having the amino acid sequence represented by the above formula (I) and having Met instead of Hse is produced as a part of the fusion protein, and then the fusion protein-producing microorganism is destroyed and To process A method for producing a polypeptide for motilin, characterized in that the individual polypeptide represented by the above formula (I) is separated from the fusion protein by the following, and fractionated and purified. 2. A DNA fragment encoding a leader sequence polypeptide portion and a motilin-like polypeptide portion is recovered by further treating the reconstructed plasmid with two types of restriction enzymes, while the plasmid pTM1 contains two types of restriction enzymes. It is cleaved by the action of an enzyme, and the motilin-like polypeptide is joined in tandem by joining the recovered DNA fragment to the breakpoint through the spacer peptide of the amino acid sequence of Gly-Ile-Phe-Met. Constructing a plasmid having the above-mentioned structure, re-cutting this plasmid if necessary, repeating the above-mentioned additional introduction operation of motilin-like polypeptide, and then incorporating into a microorganism. A method for producing the described motilin-like polypeptide. 3. An amino acid sequence of Met-Thr-Met-Ile-Thr-Asn-Ser-Gln-Gln-Gln-
Gln-Gln-Gln-Ile-Phe-Met leader sequence polypeptide followed by Phe-Val-A-Ile-Phe-Thr-Tyr-B-Glu-Leu-C
-Arg-X-Gln-Gln-Lys-Glu-Arg-D-Lys-Gly-
E-Met (A means Pro, Gly, Asn or Ser, B means Gly, P
ro means Asn or Ser, C means Gln, Glu or Asp, X means an amino acid residue other than Met, and D means Asn or Gl.
u means Asp, E means Gln, Lys or Arg) A recombinant DNA for producing a motilin-like peptide, characterized in that a motilin-like polypeptide having an amino acid sequence represented by 4. A plurality of motilin-like polypeptides in tandem, wherein the motilin-like polypeptides have a spacer peptide having an amino acid sequence of Gly-Ile-Phe-Met. A recombinant DNA for producing the motilin-like peptide according to claim (3). 5. The amino acid sequence of Met-Thr-Met-Ile-Thr-Asn-Ser-Gln-Gln-Gln-
It has a leader sequence polypeptide of Gln-Gln-Gln-Ile-Phe-Met followed by Phe-Val-A-Ile-Phe-Thr-Tyr-B-Glu-Leu-C.
-Arg-X-Gln-Gln-Lys-Glu-Arg-D-Lys-Gly-
E-Met (A means Pro, Gly, Asn or Ser, B means Gly, P
ro means Asn or Ser, C means Gln, Glu or Asp, X means an amino acid residue other than Met, and D means Asn or Gl.
u means Asp, E means Gln, Lys or Arg) Motilin-like, characterized in that a recombinant DNA in which a motilin-like polypeptide having the amino acid sequence shown by Expression plasmid for peptide production. 6. A tandem multiple motilin-like polypeptide having a spacer peptide having an amino acid sequence of Gly-Ile-Phe-Met between the motilin-like polypeptides. An expression plasmid for producing the motilin-like peptide according to claim (5).