JP2561122B2 - Functional polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to functional polypeptides, particularly human fipronectin (hereinafter abbreviated as FN)), and genes encoding them. And a genetic engineering manufacturing method using the gene.

[Conventional technology]

Fipronectin is a multifunctional glycoprotein widely distributed in human and animal blood and tissues, and is involved in physiological actions such as cell adhesion, spreading, migration, differentiation, proliferation and phagocytosis.
It is known to play an important role in tissue construction and repair, blood coagulation, and biological defense.

Recent advances in molecular biology have revealed the entire amino acid sequence and gene structure of FN [The EMBO Journal, Vol. 4, pp. 1755-1759 (198).
Five)〕.

In FN, a polypeptide having a maximum molecular weight of about 2327 and a molecular weight of about 250,000 forms a dimer with two SS bonds near the C terminus. The intramolecular amino acid sequence has a repeating structure of type I, type II, and type III, and further has binding regions (domains) for cells, collagen, heparin, fibrin, etc., independently of each other.

These functions of FN are industrially useful. For example, the cell adhesion function can be used as a coating agent for cell culture substrates or a wound healing agent. The fibrin binding capacity is
It can be considered to be used as an anticoagulant by blocking the binding between platelets and fibrin. On the contrary, a peptide having both cell adhesion activity and fibrin binding activity promotes the binding between platelets and fibrin and enhances the wound healing effect. Furthermore, recent findings have revealed that the fibrin-binding domain of FN has insulin-binding activity (46th Annual Meeting of the Japanese Cancer Society, p. 181), which shows both functions of cell adhesion activity and insulin-binding activity. The polypeptide possessed may also be used as a drug delivery system.

[Problems to be Solved by the Invention]

Thus, FN is useful as itself or as a functional fragment thereof, but it is expensive and has a limited collection amount because it is collected from blood. In addition, since there are problems such as virus infection, their use has been greatly limited.

Further, as a method for extracting a region having a specific function from FN, limited decomposition with an enzyme, limiting decomposition with promocian, etc. is known, but both are complicated in operation and the yield is very low and practical. Is hard to say.

An object of the present invention is to construct a novel functional polypeptide having both cell adhesion activity and fibrin binding activity, and to provide a method for genetically engineering the same.

[Means for solving the problem]

Briefly describing the present invention, the first invention of the present invention relates to a functional polypeptide, which is represented by the following general formula I: AB ... [I] [wherein A represents the cell adhesion activity of fibronectin]. Ala ^1235- Gln ¹⁵¹⁶ (282 amino acid residue) or Ile ^1410- Gln ¹⁵¹⁶ (107 amino acid residue) is shown as a peptide residue, and B is a fibrin-binding domain peptide residue of fibronectin, Asn ^2133- Glu ²³²⁴ ( 192 amino acid residues)
Is shown].

The second invention of the present invention relates to a gene encoding the functional polypeptide of the first invention.

The third invention of the present invention relates to a plasmid incorporating a gene encoding the functional polypeptide of the first invention.

Furthermore, a fourth invention of the present invention is an invention relating to the method for producing the functional polypeptide of the first invention, wherein the host cell into which the plasmid of the third invention is introduced is cultured,
It is characterized in that a target functional polypeptide is collected from the culture.

The present inventors have clarified the sequence of a polypeptide having an activity substantially equivalent to that of FN with regard to the FN cell adhesion-active polypeptide, developed a genetic engineering production method thereof, and applied for a patent (Patent application 63-148, and 63-31820
issue).

After that, further research was carried out, and as a result of repeated research on functional polypeptides having both cell adhesion activity and other functions, a hybrid polypeptide of cell adhesion activity polypeptide and fibrin-binding domain peptide was genetically produced. Was successful. As a result of isolating this hybrid polypeptide and examining its biological activity, it was found that it has a biological activity as a multifunctional polypeptide having both cell adhesion activity and fibrin binding activity. The present invention has been completed based on the above findings.

 Hereinafter, the present invention will be specifically described.

Means for genetically preparing a hybrid peptide of FN cell-adhesive activity polypeptide and fibrin-binding domain peptide include a plasmid containing a cell-adhesion-active polypeptide-encoding DNA and a fibrin-binding domain peptide-encoding DNA. This is accomplished by taking out the necessary fragments from the plasmid, joining the two DNA fragments together, and then ligating them into an appropriate expression vector so that their reading frames will be matched. Various DNA fragments encoding cell-adhesive active polypeptides have already been cloned (Japanese Patent Application Nos. 63-148 and 63-63).
−31820). The required part can be excised from these plasmids.

On the other hand, the DNA fragment encoding the fibrin domain is p
PLF2435 [Biochemistr, which was constructed by connecting the FN cDNA parts of LF5, pLF3, pLF4 and pLF2, was constructed.
y) Vol. 25, pp. 4936-4941 (1986)] and can be prepared by cutting out the required fragment.

The hybrid peptide can be expressed in Escherichia coli by connecting a DNA fragment encoding a hybrid peptide of a cell adhesion-active polypeptide and a fibrin domain polypeptide to an appropriate expression vector and introducing it into Escherichia coli. As the expression vector, all existing vectors can be used.

Confirmation of the hybrid peptide by Escherichia coli can be examined by immunoplating. Whole cell protein
After separation by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), the migration pattern is transferred to a nitrocellulose filter and reacted with both a monoclonal antibody that recognizes FN cell-adhesion domain and a monoclonal antibody that recognizes the fibrin domain. Bands can be detected.

Conventional chromatographic techniques are used to purify the expressed peptides. For example, bacterial pellets are suspended in buffer and sonicated to obtain a soluble fraction. This is applied to a column of Sepharose 4B to which the antibody used for immunoblotting is bound to perform affinity purification. By collecting the target fraction by immunoblotting, a functional polypeptide having both cell adhesion activity and fibrin binding activity can be obtained. If necessary, it can be further purified by FPLC or HPLC.

The functional polypeptide thus obtained is used for measuring biological activities such as cell adhesion activity, fibrin binding activity and insulin binding activity.

NRK cells were used to measure the cell adhesion activity, for example,
The sample is dissolved in buffer and adsorbed on a microplate, NRK cells are added, and incubation is performed at 37 ° C for a certain period of time. The strength of cell adhesion activity can be shown by observing cell spreading under a microscope and comparing the minimal amount required for expression of spreading with native FN. Fibrin-bound Sepharose 4B was used to measure fibrin-binding activity.
[Reference: Thrombosis Researc
h) Vol. 2, pp. 137-154 (1973)] can be used. That is, after passing the sample through the fibrin-binding column, the passing solution is subjected to SDS-PAGE to determine the presence or absence of fibrin-binding activity. The insulin-binding activity can be determined according to the immunoblotting technique. That is, after separating the sample by SDS-PAGE, it is transferred to a nitrocellulose filter and treated with enzyme-labeled insulin. After washing the filter, bound insulin can be determined by enzymatic activity.

Thus, a preferable example of the obtained peptide is Ala ¹²³⁵ -Gln ¹⁵¹⁶ (282) which has the cell adhesion activity of FN.
Amino acid residue peptide) or Ile ¹⁴¹⁰ -Gln ¹⁵¹⁶ (107
And a fibrin-binding domain peptide Asn ^2133- Glu ²³²⁴ (192 amino acid residues).

The amino acid sequence of the polypeptide having the cell adhesion activity of FN is as follows: Also, the amino acid sequence of the fibrin-binding domain peptide of FN described above is as follows: [Examples] Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited to these examples.

Example 1 FN cell adhesion domain Ile ¹⁴¹⁰ -Met ¹⁵¹⁷ (108 amino acid residues) and fibrin binding domain Asn ²¹³³ -Glu ²³²⁴
Cloning of cDNA fragment encoding (192 amino acid residues) (see FIG. 1) FIG. 1 is a process drawing of the construction of pTF1301 according to the present invention.

(1-1) Preparation of cDNA fragment FN cell adhesion drain Ile ¹⁴¹⁰ -Met ¹⁵¹⁷ (108 amino acid residues) A 7.8 kb plasmid pTF201 (Japanese Patent Application No. 63-148) containing a cDNA fragment encoding a restriction enzyme. Xba I buffer, 100 unit Xba I and 100 units Eco
Incubation was performed at 37 ° C. for 2 hours in 100 μl of a reaction solution containing RI. The reaction solution was used as a HPLC column manufactured by DIAGEN, and Nucleogen DEAE-4000 (6
X 125 mm) to obtain 2 μg of 0.41 kb fragment. Next, this fragment was treated with 20 units of Fck I for 1 hour at 37 ° C., and Xba I-Fck I fragment (180 bp) and Fok I-Fok I were prepared by the same purification method.
250 ng of each fragment (203 bp) was obtained.

On the other hand, the fibrin-containing domain Asn ²¹³³ −Glu ²³²⁴ (19
5.9 kb plasmid pLF2435 containing a cDNA fragment encoding 2 amino acid residues) [Biochemistry Vol. 25, 4936
~ 4941 (1986)] 100 µg of buffer for Hind III, 1
Includes 00 unit Hind III and 100 unit Hinc II
Incubation was performed at 37 ° C. for 2 hours in 100 μl of the reaction solution. This was subjected to agarose electrophoresis to cut out a 1.2 kb fragment (yield 2 μg). 1 μg of this fragment was added to Sau3A I 3
The mixture was treated at 7 ° C. for 1 hour and applied on a Nucleodiene DEAE-4000 column to obtain 200 ng of Hinc II-Sau3A I fragment (621 bp).

(1-2) Preparation of Synthetic DNA Adapter An adapter (chain lengths 24 and 20, see FIG. 1) for connecting the cDNA fragments of the cell adhesion domain and the fibrin-binding domain was applied using a DNA synthesizer manufactured by Applied Biosystems. Synthesized to obtain 2.7 μg and 2.3 μg, respectively. 100 ng of each was made into a double chain by an annealing operation.

(1-3) Ligation of cDNA fragment and vector The cDNA fragment obtained in (1-1) is the secretory expression vector pIN.
III-omp AI [Jembo Journal, Volume 3, 243
7-2442 (1984)].

That is, 10 ng of Xba I-Fok I fragment (180 bp), Fok I-
Fok I fragment (203bp) 10ng, Hinc II-Sau3A I fragment (621b
p) 30 ng, 10 ng of the synthetic DNA adapter obtained in (1-2), and pIN III-om in which the Xba I-BamH I insert fragment was previously removed.
50 ng pA I vector with 0.5 mM A buffer for T ₄ DNA ligase
Contains P, 10 mM DTT and 2.8 units of T ₄ DNA ligase 20
Incubation was performed overnight at 16 ° C. in μl of the reaction solution. This reaction solution was used for transformation of Escherichia coli.

(1-4) Transformation of Escherichia coli and Confirmation of Plasmid Using 20 μl of the reaction solution obtained in (1-3), Escherichia coli HB101
Was transformed. The 21 clones of the obtained transformants were analyzed for plasmids. That is, each clone was cultured with shaking in 1.5 ml of L-broth containing 50 μg / ml ampicillin overnight, a plasmid was prepared by the rapid method, and a part thereof was double-digested with Xba I and Sal I. This was separated by agarose electrophoresis and the expected Xba I-Xba
The generation of bands for the I fragment (0.56 kb) and the Xba I-Sal I fragment (1.5 kb) was examined. As a result, the band of interest was recognized in 12 clones. Furthermore, the nucleotide sequence was determined by the dideoxy method, and it was confirmed that the nucleotide sequence had the correct nucleotide sequence except that the c-terminal methionine codon of 108 amino acids in the cell adhesion domain was deleted.

This recombinant plasmid was named pTF1301. In addition, Escherichia c
It was labeled as oli HB101 / pTF1301 and deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology.
47)].

Example 2 FM cell adhesion domain Ala ¹²³⁵ -Gln ¹⁵¹⁶ (282 amino acid residues) and fibrin binding domain Asn ²¹³³ -Glu
Cloning of cDNA fragment encoding ²³²⁴ (192 amino acid residues) (see FIG. 2) FIG. 2 is a process drawing of the construction of pTF1801 according to the present invention.

(2-1) Preparation of Xba I-Pvu II fragment of pTF901 cDNA encoding cell adhesion domain Ala ¹²³⁵ -Met ¹⁵¹⁷
Fragment-containing plasmid pTF901 (Japanese Patent Application No. 63-148) 20μ
g containing 50 units of Xba I and 50 units of Pvu II 2
Incubation was carried out at 37 ° C. for 1 hour in 00 μl of the reaction solution.
This reaction solution was subjected to agarose electrophoresis, and Xba I-Pvu I
The I insert fragment (0.61 kb) was cut out (yield 400 ng).

(2-2) Preparation of Xba I-Pvu II fragment of pTF1301 25 μg of the plasmid pTF1301 obtained in Example 1 was incubated at 37 ° C. for 1 hour in a 200 μl reaction solution containing 50 units of Xba I and 50 units of Pvu II. did. This reaction solution was subjected to agarose electrophoresis to cut out the Xba I-Pvu II insert fragment (0.40 kb) (yield 400 ng).

In addition, a 8.0 kb pector lacking the Xba I-Xba I insert fragment was simultaneously excised (yield 2 μg), dephosphorylated, and used for cloning.

(2-3) Ligation of Xba I-Pvu II fragment (0.61 kb and 0.40 kb) 400 ng of 0.61 kb fragment obtained in (2-1) and 400 ng of 0.40 kb fragment obtained in (2-2) were combined with T ₄ DNA ligase. Buffer, 0.5m
Incubation was carried out for 3 hours at 16 ° C. in 20 μl of a reaction solution containing M ATP, 10 mM DTT and 2.8 units of T ₄ DNA ligase.
The reaction was stopped by treatment at 65 ° C for 10 minutes, and the buffer was removed with Xba I
Optimal conditions, 10 units of Xba I added, 37 ℃, 2
Allowed to react for hours. The reaction solution is subjected to agarose electrophoresis,
The XbaI-XbaI fragment (1.01 kb) was cut out (yield 20 ng).

(2-4) Ligation of Xba I-Xba I fragment (1.01 kb) and vector 20 ng of Xba I-Xba I fragment (1.01 kb) obtained in (2-3) and 8.0 kb obtained in (2-2) the vector 20 ng T ₄ DNA ligase buffer were, 0.5 mM ATP, in 10 mM DTT and 2.8 Yunitsuto
Incubation was performed for 3 hours at 16 ° C. in 20 μl of a reaction solution containing T ₄ DNA ligase. This reaction solution was used for transformation of Escherichia coli.

(2-5) Transformation of E. coli and confirmation of plasmid Escherichia coli HB101 was prepared by using 20 μl of the reaction solution obtained in (2-4).
Was transformed. The plasmids were confirmed for 12 clones in the obtained transformants. The plasmid prepared by the rapid method was double-digested with Xba I-Pvu II and subjected to agarose electrophoresis to examine the generation of expected insert fragments (0.61 kb and 0.40 kb). As a result, the band of interest was recognized in one clone. For this clone,
When oRI digestion was performed and the orientation of the insert was examined, it was confirmed that the insert was in the correct orientation. In addition, the nucleotide sequence was determined by the dideoxy method, and it was confirmed that the target sequence was included.

This recombinant plasmid was named pTF1801. In addition, Escherichia coli HB101 harboring this plasmid was
Labeled as ia coli HB101 / pTF1801 and deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology
−9948)].

Example 3 FN chimeric polypeptide Ile ¹⁴¹⁰ -Gln ¹⁵¹⁶ / Asn ²¹³³ -Gl
Production and purification of u ²³²⁴ (299 amino acid residues) The HB101 / pTF1301 obtained in Example 1 was inoculated into two test tubes containing 5 ml of L-broth supplemented with 50 μg / ml of ampicillin and shaken at 37 ° C. overnight. Cultured. This was inoculated into a 2 liter Erlenmeyer flask containing 500 ml of the same medium and the culture was continued.When the absorbance at 660 nm reached 0.3, 2 mM IPTG was added.
(Isopropyl β-thiogalactoside) was added, and the cells were collected after 20 hours. Whole cell pellets were washed with 10 mM Tris-HCl (p
H7.5), suspended in a solution containing 5 mM EDTA, sonicated and centrifuged at 12000 rpm for 30 minutes to obtain 40 ml of the supernatant. This was passed through a Sepharose 4B column (8 ml) to which an anti-FN monoclonal antibody (FN-12, Takara Shuzo) was bound. The column was washed with Wash Buffer A (20 mM Tris HCl, pH 7.5), and then Wash Buffer B (20 mM Tris HCl, pH 8.0, 0.
It was washed with 1MKSl). Finally, elution was performed with an elution buffer (50 mM glycine HCl, pH 2.3, 0.2 M KCl). Eluted fractions were collected and electrophoretically almost single 34kd polypeptide 500μg
I got

Example 4 FN chimeric polypeptide Als ¹²³⁵ -Gln ¹⁵¹⁶ / Asn ²¹³³ -Gl
u ²³²⁴ The HB101 / pTF1801 obtained in Production and Purification Example 2 of (474 amino acid residues) and 2l cultured in the same manner as in Example 3, was purified by anti-FN monoclonal antibody (FN-12) Sepharose 4B column. This antibody column eluate fraction contained, in addition to the target polypeptide of 55 kd, a degradation product of the polypeptide of 35 kd. There it was subsequently purified on a Fibrin-Sepharose 4B column.
That is, the antibody column elution fraction was passed through a fibrin-sepharose 4B column (10 ml) prepared by the method of DL Heene et al. [Trombosis Research, Vol. 2, pp. 137-154 (1973)]. The column was washed with a washing buffer (10 mM Tris HCl, pH 7.6, 50 mM NaCl, 0.5 mM EDTA) and then eluted with an elution buffer (25 mM Tris HCl, pH 7.6, 6 M urea). The eluted fractions were collected and electrophoretically separated.
100 μg of 55 kd polypeptide was obtained.

Example 5 Measurement of biological activity Chimeric polypeptide Ile ¹⁴¹⁰ -Gl obtained in Examples 3 and 4
n ¹⁵¹⁶ / Asn ²¹³³ -Glu ²³²⁴ (107CBP / 192FBP, 299 amino acid residues), Ala ¹²³⁵ -Gln ¹⁵¹⁶ / Asn ²¹³³ -Glu ²³²⁴ (282CBP / 192FBP,
The following biological activities were measured using 474 amino acid residues).

(5-1) Cell adhesion activity Ruoslahti et al. [Methods in Enzymology]
Cell adhesion activity was measured according to Vol. 82, pp. 803-831 (1981)]. That is, the sample was serially diluted with physiological saline or distilled water, and 50 μl thereof was dispensed into a 96-well microplate and incubated overnight at 4 ° C. to attach the sample to the plate. Next, the plate was washed twice with PBS buffer, 100 μl of 3% BSA was added and incubated at 37 ° C. for 1 hour to float the plate. After washing the plate twice with PBS buffer, the rat kidney cells (NRK-49F) suspended in the Eagle's minimum medium (MEM) at 10 ⁶ cells / ml were divided at 100 μl / well. Note, 37
Incubated at ℃ for 2-3 hours.

The NRK-49F cells used were those that had been cryopreserved and then pre-cultured and then trypsinized.

Cell spreading is observed under a microscope, and the minimum amount required for spreading is shown as the minimum cell adhesion activity. The results are shown in Table 1 below together with other activities.

Polypeptides having only cell adhesion domains of 108 amino acid residues and 283 amino acid residues [108CBP and 283CBP (Il
e ¹⁴¹⁰ -Met ¹⁵¹⁷ and Ale ¹²³⁵ -Met ¹⁵¹⁷ ) Japanese Patent Application No. 63-148
No.] was measured at the same time and compared with the chimeric polypeptide.

As a result, 107CBP / 192FBP was about 52 times more active than 108CBP. 282CBP / 192FBP was found to have the same activity as 283CBP.

(5-2) Fibrin binding activity Sekiguchi et al. [Journal of Biological
Chemistry (Journal of Biological Chemistry) 2nd
56, 6452-6462 (1981)].
The fibrin binding activity was examined by adsorption on Sepharose 4B column. The method of DL Hane et al. [Trombosis
Research, Vol. 2, pp. 137-154 (1973)], the chimeric polypeptide Ile ¹⁴¹⁰ -Gln ¹⁵¹⁶ / Asn ²¹³³ -Glu ^{2324 was} added to the fibrin-sepharose 4B column (1.4 ml).
5 μg of (107 CBP / 192 FBP, 299 amino acid residues) was passed.
7.5 ml wash buffer (10 mM Tris HCl, pH 7.6, 50 mM
After removing the non-adsorbed fraction with NaCl, 0.5 mM EDTA), the adsorbed fraction was eluted with an elution buffer (25 mM Tris HCl, pH 7.6, 6 M urea). Fraction each 0.5ml, 107CBP / 192FBP
The elution position of was examined by an ELISA method using an anti-FN monoclonal antibody (FN-12, Takara Shuzo). As a result, 107 CBP /
192FBP was found in the adsorbed fraction and was confirmed to have fibrin-binding activity. Similar results were obtained with Ala ¹²³⁵ −Gln ^1516.
/ Asn ²¹³³ / Glu ²³²⁴ (282CBP / 192FBP, 474 amino acid residues)
But it was confirmed. Further, Ile ¹⁴¹⁰ -Met ¹⁵¹⁶ (108CBP) having no fibrin binding domain and Ala ¹²³⁵ -Met ¹⁵¹⁶
(283CBP) was not adsorbed to fibrin-sepharose 4B. (See Table 1) (5-3) Insulin binding activity It is known that the fibrin binding domain of FN has insulin binding activity (The 46th Annual Meeting of the Japanese Cancer Society, p.181).

Therefore, the insulin binding activity of the chimeric polypeptide was examined. 107CBP / 192FBP, 282CBP / 192FBP, 108CBP, 283CBP
Bi-Rad B manufactured by serially diluting 1/2 from 20 μg
It was adsorbed on a nitrocellulose membrane using IO-DOT ^™ .
This nitrocellulose membrane was subjected to prototyping operation with a PBS buffer containing 3% BSA, and then left at room temperature for 2 hours in a PBS buffer containing 50 μg / ml peroxidase-labeled insulin (Shizuma). Next, this nitrocellulose membrane was washed twice with PBS for 5 minutes, and the bound peroxidase-insulin was detected using 4-chloro-1-naphthol and hydrogen peroxide as substrates. As a result 108C
In BP and 283CBP, no color is seen, but 10
7CBP / 192FBP and 282CBP / 192FBP showed color development corresponding to the concentration. Therefore, 107CBP / 192FBP and 282CBP / 19
It was confirmed that 2FBP has insulin-binding activity (see Table 1).

[Effects of the Invention] As described in detail above, according to the present invention, a functional polypeptide having at least a cell adhesion activity and a fibrin binding activity, a gene encoding them, and a gene encoding the functional polypeptide, Provided is a genetically engineered production method capable of mass-producing a sex polypeptide.

The above-mentioned polypeptide is useful for various applications such as wound treatment and drug delivery system.

[Brief description of drawings]

1 and 2 are process diagrams for constructing the plasmid of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:19) (72) Inventor Yuki Sugawara 3-4-1 Seta, Otsu City, Shiga Prefecture Central Research Laboratory, Sake Brewing Co., Ltd. (72) Ikunoshin Kato, 3-4-1, Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Taka Brewing Co., Ltd. (56) Reference JP-A-62-89699 (JP, A) ) JP-A-62-272975 (JP, A) JP-A-62-282593 (JP, A) JP-A-62-236485 (JP, A) JP-A-62-82197 (JP, A)

Claims (4)

(57) [Claims] 1. A compound represented by the following general formula I: AB ... [I] [wherein A is a peptide residue having fibronectin cell adhesion activity, and is represented by Ala ¹²³⁵ -Gln ¹⁵¹⁶ (282 amino acid residue) or Ile ^1410. -Gln ¹⁵¹⁶ (107 amino acid residues), B is the fibrin-binding domain peptide residue of fibronectin, Asn ²¹³³ -Glu ²³²⁴ (192 amino acid residues)
] A functional polypeptide represented by the formula 2. A gene encoding the functional polypeptide according to claim 1. 3. A plasmid incorporating a gene encoding the functional polypeptide according to claim 2. 4. The functional polypeptide according to claim 1, wherein the host cell introduced with the plasmid according to claim 3 is cultured, and the functional polypeptide according to claim 1 is collected from the culture. Manufacturing method.