WO1996003503A1

WO1996003503A1 - Process for producing urinary trypsin inhibitor and domains thereof, novel polypeptide related to the both, and process for producing the polypeptide

Info

Publication number: WO1996003503A1
Application number: PCT/JP1995/001449
Authority: WO
Inventors: Shoji Ideno; Takashi Goto; Hajime Horii
Original assignee: The Green Cross Corporation
Priority date: 1994-07-21
Filing date: 1995-07-21
Publication date: 1996-02-08

Abstract

A process for producing a urinary human trypsin inhibitor (UTI) and domains thereof by using a yeast of the genus Pichia as the host cell; an expression system related thereto; a novel polypeptide at least having the amino acid sequence represented by formula (I) in the specification; a DNA coding for the same; an expression vector containing the DNA; a transformant containing the vector; and a process for producing the polypeptide. The above UTI, expression system of each domain thereof, and process for producing the UTI and domains thereof enable UTI and domains thereof to be mass-produced readily and efficiently. The use of the expression system facilitates the preparation of variants or derivatives of UTI and so forth and contributes to the development of novel medicines such as a protease inhibitor. The invention also provides a novel useful polypeptide which is more excellent in elastase inhibitory activity than natural UTI and can be mass-produced.

Description

Specification

Method for producing urinary trypsin inhibitor Yuichi and its domain, and novel polypeptides related thereto and method for producing the same

〔Technical field〕

The present invention relates to a method for producing a urinary trypsin inhibitor (hereinafter, simply referred to as UTI) and a Kunitz-type domain thereof by a genetic engineering technique using Pichia yeast as a host cell. Furthermore, the present invention provides a novel polypeptide having excellent protease inhibitory activity, a DNA encoding the polypeptide, a vector having the DNA, a transformant transformed with the vector, and culturing the transformant. And a method for producing the novel polypeptide.

(Background technology)

In inflammation typified by acute inflammation, various site forces, such as interleukin-16, interleukin-18, and tumor necrosis factor (TNF), are induced by bacterial infection associated with soil inflammation in local tissues of the inflammation. As a result, migration and activation of neutrophils in tissues and organs occurs. Such conditions cause disseminated intravascular coagulation (DIC) and multiple organ failure (MOF), which can cause severe symptoms. The characteristic features of the transition from the occurrence of local inflammation to systemic disease states such as MOF and DIC are (1) the progression of the disease state from the local to the whole body rapidly progresses in a short time, (2) the final There are two points that involve the involvement of proteases such as elastase (HNE) released from activated neutrophils and elastase (HPE) released from skeletal cells. Therefore, in the treatment of otitis, etc., two approaches are considered: suppressing the progression in the early stage of inflammation and inhibiting the protease. However, in the former approach, it is difficult to develop an effective drug because of the involvement of multiple factors and the progress is rapid.At present, the latter approach is considered effective, including new development. I have.

From this viewpoint, at present, low-molecular-weight synthetic protease inhibitors and proteinaceous proteinase inhibitors, which are biological components, are used in the treatment of inflammation represented by spleenitis. However, most of the current products have elastase inhibitory activity. The development of a protease inhibitor having an elastase inhibitory activity has been desired.

Among the main proteinaceous proteases, those having elastase inhibitory activity include α-antitrypsin (1-Α) and UTI, all of which mainly inhibit neutrophil elastase.

al-AT is a serine protease inhibitor present in a large amount in blood and has a strong inhibitory activity on neutrophil elastase, but is known to be easily inactivated by active oxygen.

On the other hand, it is known that the elastase inhibitory activity of UTI is weaker than that of cH-AT. Also, since UTI is a protein purified from human urine, it is difficult to secure raw materials when mass-producing it, and it is also difficult to produce a formulation with a constant component because the raw materials are heterogeneous. . In addition, problems common to general preparations derived from biological components, such as contamination with unknown components and human infectious viruses, must be considered. Furthermore, there has been no report of producing UTI and its domains and novel polypeptides related thereto in large quantities by genetic techniques, that is, there is no known polypeptide molecule that can be produced in large quantities by genetic engineering techniques. Absent.

Thus, there are many problems to be solved in protein protease inhibitors currently used as pharmaceuticals. Therefore, in order to enable the treatment of a disease associated with in vivo C protease, a method for mass-producing a useful proteinase inhibitor that inhibits the protease should be established, and a novel method that has excellent protease inhibitory activity. Material development is needed.

[Disclosure of the Invention]

An object of the present invention is to provide a new method for producing UTI and its respective domains by genetic engineering techniques, especially using Pichia yeast as a host cell. Another object of the present invention is to provide a useful novel polypeptide having a strong neutrophil elastase inhibitory activity and preferably capable of being produced in a large amount by a genetic engineering technique, and a method for producing the same by the genetic engineering technique. As a result of intensive studies to achieve the above object, the present inventors succeeded in cloning the cDNA of UTI, and expressing and producing UTI and each domain using Pichia yeast as a host cell. We have established a method for producing E. coli by genetic engineering. Furthermore, a novel borohydride having improved neutrophil elastase inhibitory activity, which is a disadvantage of natural UTI, having a stronger neutrophil elastase inhibitory activity, and preferably also having a trypsin inhibitory activity. As a result of various studies for the purpose of producing butide, it was found that the amino acid-substituted UTI obtained by substituting a part of the amino acid of UTI and the shortened UTI having only the active domain have the above-mentioned useful properties. The present inventors succeeded in producing a large amount of the protein by genetic engineering and completed the present invention.

That is, the outline of the present invention is as follows.

(1) an expression vector for a yeast belonging to the genus Pichia containing DNA having a nucleotide sequence encoding the amino acid sequence of Kunitz type domain 1 of UTI, having a nucleotide sequence encoding the amino acid sequence of Kunitz type domain 2 of UTI An expression vector for Pichia yeast containing DNA, and an expression vector for Pichia yeast containing DNA having a base sequence encoding the amino acid sequence of UTI.

(2) A yeast belonging to the genus Pichia transformed with any of the expression vectors for Pichia 厲 yeast according to (1).

(3) culturing the yeast belonging to the genus Pichia according to (2) to produce a protein having at least one amino acid sequence of a Knitz-type domain of UTI, and collecting the protein from the resulting culture. A method for producing a protein having at least one kind of Kunitz-type domain amino acid sequence of UTI, characterized by comprising:

(1) A novel polypeptide having at least the amino acid sequence represented by the formula (I). O 96/03503

Cys-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-Cys-ile-Ala-Phe-Phe-Pro-Arg-Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-Met-Ala -Cys-Glu-Thr-Phe-Gln-Tyr-Gly-Gly-Cys-Met-G1y-Asn-Gly-Asn-Asn- (Formula I)

Phe-Val-Thr-Glu-Lys-Glu-Cys-Leu-Gln-Thr-Cys

The novel polypeptide may have an amino acid sequence represented by the formula:

Thr-Val-Ala-Ala-Cys-Asn-Leu-Pro-Ile-Val

Arg-G 1 y-Pro-Cys-Arg-Ala-Phe- I le-Gl n-Leu-

Trp-Ala-Phe-Asp-Ala-Val-Lys-Gly-Lys-Cys-

Va 1 -Leu-Phe-Pro-Tyr-G 1 y-G 1 y-Cys-Gl n-G 1 y-

Asn-Gly-Asn-Lys-Phe-Tyr-Ser-Glu-Lys-Glu- (Formula ΠI)

Cys-Arg-G 1 u-Tyr-Cys-C 1 y-Val-Pro-G 1 -As p-

Gly-Asp-Glu-Glu-Leu-Leu-Arg-Phe As the novel polypeptide, a polipeptide having an amino acid sequence of the following formula IX is preferably used.

Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr-Ser-

Ala-Gly-Pro-Cys-I le-Ala-Phe-Phe-Pro-Arg-

Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-Met-Ala-Cys-

Glu-Thr-Phe-Gln-Tyr-Gly-Gly-Cys-Met-Gly-

Asn-G 1 y-Asn-Asn-Phe-Va 1 -Thr-Glu-Lys-G 1 ir-

Cys-Leu-Gln-Thr-Cys-Arg-Thr-Val-Ala-Ala-

Cys-Asn-Leu-Pro-I le-Val-Arg-Gly-Pro-Cys-

Arg-A 1 a-Phe- I le-Gl n-Leu-Trp-Ala-Phe- Asp-

A 1 a-Va 1 -Lys-G 1 y-Lys-Cys-Val-Leu-P e-Pro-

Tyr-Gly-G1y-Cys-G1n-Gly-Asn-G1y-Asn-Lys-

Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu-Tyr- (Formula)

Cys-Gly-Val-Pro-Gly-Asp-Gly-Asp-Glu-Glu-

Leu-Leu-Arg-Phe In addition, these polypeptides may have an amino acid sequence represented by Formula I [N-terminal].

A 1 a-Va 1-Leu-Pro-Gln-GluGlu-Glu-Gly-Ser-Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr- (Equation ¹¹ )

And ys is preferably a novel polypeptide having an amino acid sequence represented by Formula IV. Ala-Val-Leu-Pro-Gln-Glu-Glu-Glu-Gly-Ser-

Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr-

Lys-Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr-

Ser-Ala-G 1 y-Pro-Cy s- 11 e- Ala-Phe-Phe-Pro-

Arg-Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-et-Ala-

Cys-Glu-Thr-Phe-Gln-Tyr-Gly-Gly-Cys-Met-

Gly-Asn-Gly-Asn-Asn-P e-Val-Thr-Clu-Lys-

Glu-Cys-Leu-Gln-Thr-Cys-Arg-Thr-Val-Ala-

Ala-Cys-Asn-Leu-Pro-Ile-Val-Arg-Gly-Pro-

Cys-Arg-Ala-Phe-Ile-Gln-Leu-Trp-Ala-Phe-

Asp—Ala_Val—Lys—Gly—Shys—Cys—Val—Sheu—Phe—

Pro-Tyr-Gly-Gly-Cys-Gln-Gly-Asn-Gly-Asn-

Lys-Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu- (Formula IV)

Tyr-Cys-Gly-Val-Pro-Gly-Asp-Gly-Asp-Glu-

Glu-Leu-Leu-Arg-Phe

(5) The novel polypeptide according to (4), which has an inhibitory activity on neutrophil elastase.

(6) The novel polypeptide according to (4), which has an inhibitory activity on tribcine and has an enhanced inhibitory activity on neutrophil elastase as compared with natural UTI.

(7) A DNA having a base sequence encoding the novel polypeptide according to any one of (4) to (6).

(8) A vector comprising the DNA according to (7).

(9) A transformant transformed with the vector of (8).

(10) A method of culturing the transformant of (9) to produce the novel polypeptide of (4) to (6), and collecting the novel polypeptide from the obtained culture. (4) The method for producing a novel polypeptide according to (6). (11) Transformation with a vector having a nucleotide sequence encoding the amino acid sequence of the following formula (II) linked to the 5 'end of the nucleotide sequence encoding the amino acid sequence containing the active site of the protease inhibitor: A method for enhancing the expression of protease inhibitor Yuichi, comprising culturing a transformed transformant.

Ala-Va 1-Leu-Pro-Gln-Glu-Gl uG 1 uG 1 y-Ser- Gly-Gly-Gly-Gl n-Leu-Va 1 -Thr-G 1 u-Va 1 -Thr- ⁽ Equation ^Π )

Lys

[Brief description of drawings]

FIG. 1 shows the primary structure of UTI isolated from urine (Watcher. E and Hochstrasser. K., Hoppe-Seyler's Ζ. Physiol. Chem., 362, 1351, 1981) ο

FIG. 2 is a diagram showing the primary structure of the Kunitζ type domain 1 and the Kunitz type domain 2 of UTI.

FIG. 3 is a diagram showing the nucleotide sequence (120 to 240) of the cloned UTI cDNA. The base number was in accordance with the report of J. F. Kaumeyer et al., Nucl. Acads Res. 14 (20), 7839-7850 (1986).

FIG. 4 is a diagram showing the nucleotide sequence (241 to 540) of the cloned UT1 cDNA. The base number was determined according to the report of J. F. Kaumeyer et al., Nucl. Acads Res. 14 (20), 7839-7850 (1986). Note that the restriction enzyme sites in the figure are those used in each Example.

FIG. 5 is a diagram showing the base sequence (541-780) of the cloned UTI cDNA. The base number was in accordance with the report of J.F. Kaumeyer et al., Nucl. Acads Res. 14 (20), 7839-7850 (1986). The amino acid sequence determined from UTI cDNA is shown below. The restriction enzyme sites shown in the figures are those used in each Example.

Figure 6 shows the nucleotide sequence of the cloned UTI cDNA (781-1020). FIG. The base number was in accordance with the report of F. Kaumeyer et al., Nucl. Acads Res. 14 (20), 7839-7850 (1986). The amino acid sequence determined from the UTI cDNA is shown below. The restriction enzyme sites shown in the figures are those used in each Example. FIG. 7 is a view showing the base sequence (1021-1235) of the cloned UTI cDNA. The base number was in accordance with the report of JF Kaumeyer et al., Nucl. Acads Res. 14 (20). 7839-9850 (1986). The amino acid sequence determined from the cDNA of UTI is shown below. The restriction enzyme sites in the figures are those used in each example. FIG. 8 is a view showing a modified base sequence of a region encoding the N-terminus and a region encoding the C-terminus of UTI.

FIG. 9 is a diagram showing a construction process of # 7_BbeI—ERI.

FIG. 10 is a diagram showing the process of constructing pUT I-N from Bbe I—ER I # 7.

Fig. 11 is a diagram showing the process of constructing pUT I-C from # 7ZBb e I-ER I.

FIG. 12 is a diagram showing a construction process of pUTINC.

FIG. 13 is a diagram showing the steps of constructing pUTIN-CZERI.

FIG. 14 is a diagram showing a restriction map of pUTIN-CZERI and pA〇807N, and a process for constructing pHH310.

FIG. 15 shows the nucleotide sequence of the SUC2 signal.

FIG. 16 is a diagram showing the nucleotide sequence of a synthetic DNA that complements the region encoding the C-terminus of Kunitz type domain 1 of UTI.

FIG. 17 is a diagram showing the nucleotide sequence of a synthetic DNA that complements a region encoding the N-terminus of Kunitz type domain 2 of UTI.

FIG. 18 is a diagram showing the gene unit of the N-terminal butyl-added K unitz type domain 1 and the Kunitz type domain 2 of UTI. In the figure, the domain of each domain is indicated by the amino acid number of UTI. FIG. 19 is a diagram showing a construction process of pHH305 and a restriction enzyme map thereof. FIG. 20 is a diagram showing a restriction enzyme map of pHH313.

FIG. 21 is a diagram showing a construction step of pHH306 and a restriction enzyme map thereof. FIG. 22 is a diagram showing a restriction enzyme map of pHH314.

FIG. 23 is a view showing a profile of HPLC-GPC of purified Intact-rUTI.

FIG. 24 is a view showing a profile of HP LC-GPC of purified rD2. FIG. 25 is a diagram showing a region used for mutagenic primer.

FIG. 26 is a diagram showing a construction step of pHH334.

FIG. 27 shows the effect of chloramine T oxidation of Ep1-UTI and native UTI on neutrophil elastase inhibition.

Figure 28 shows the synthetic DNA corresponding to the region from the 5 'end to the Ec052 I site that encodes the 21 amino acid region added to the N-terminal side of the Kunitz type domain 1 of UTI. It is a figure which shows a base sequence. In the figure, the bases enclosed in squares are bases that have been replaced due to disappearance of the Pvull site.

FIG. 29 is a diagram showing a construction process of pHH336 and a restriction map thereof. FIG. 30 is a diagram showing a construction step of pHH339 and a restriction enzyme map thereof.

[Detailed description of the invention]

Hereinafter, the present invention will be described in detail. The abbreviations used in the present specification and the drawings are based on commonly used abbreviations in the relevant field.

First, the UTI and its Knitsz-type domain according to the present invention will be described. UTI was initially isolated from human urine according to the method of Proksch (Proksch G. J., et al., Lab. Clin. Med., 79, 491-499, 1972). It is a proteinase inhibitor that has been studied (Sumi et al., Japanese Journal of Physiology, Vol. 39, pp. 53-58, 1977) and has the primary structure shown in Fig. 1 by studies by Watcher, E. Known (Watcher, E and Hochstrasser, K., Hoppe-Seyler's

Physiol. Chem., 362. 1351. 1981). The primary structure of the protein shown in FIG. 1 is the amino acid sequence directly obtained from the purified protein.

The UTI is continuous from two structurally related Kunit I type domains. In other words, in UTI, one Knitz-type domain 1 is located in the N-terminal region of UTI and is defined as amino acid residues 22 to 77 counted from the N-terminal, and the other Knitzitz domain 1 Type domain 2 is located in the C-terminal region of UTI, and is defined as amino acid residues 78 to 1443 counted from the N-terminus (Hochstrasser, et al., Hoppe-Seyler's Z. Physiol Chem., 362, 1351, According to the DNA recombination method (the amino acid sequence is deduced from the cDNA sequence), the Kunitz-type domain 1 and Kunitz-type domain 2 of UTI referred to in the present invention are each. And has been reported to have the amino acid sequence shown in Fig. 2 [JF Kaum eyer et al., Nucl. Acids Res. 14 (20), p7844-7845. 1986: Amino acid numbers 86, 87 and 138 are from proteins. This differs from the directly determined amino acid sequence (Fig. 1).] Kunitz-type domain 1 and Kunitz-type domain referred to in the present invention. The term “in 2” substantially means each of these domains, and according to this DNA recombination method, the UTI structural gene is composed of 441 bases and encodes 147 amino acids. No sequence with a terminal amino acid at position 147 in UTI has been reported. (Hoc strasser, K. et al., Hoppe-Seyler's Z. Physiol. Chem., 362, 1351, 1981). Therefore, when expressing UTI by genetic engineering, it is considered preferable to use a sequence found in nature.In the present invention, the amino acid at position 145 is replaced with the C-terminal amino acid of K unitz type domain 2. And

In addition, K unitz type domain 1 and K unitz type domain 2 have different specific protease inhibitory activities, respectively, edited by CGebhard & Hochstraber, Proteinase Inhibitors, Barrett & Salvesen, Elsevier ^ 1986, p. 375) _The present invention relates to an expression vector containing a DNA having a base sequence encoding the amino acid sequence of the Kunitz-type domain 1 of UTI, a nucleotide sequence encoding the amino acid sequence of the Kunitz-type domain 2 of UTI. And an expression vector containing a DNA having a base sequence encoding the amino acid sequence of UTI.

The amino acid sequence of UTI specifically refers to an amino acid sequence represented by the following formula V:

Ala-Val-Leu-Pro-Gln-Glu-Glu-Glu-Gly-Ser- Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr-Lys-Lys-Glu-Asp-Ser- Cys-Gln-Leu-Gly-Tyr- Ser-Ala-Gly-Pro-Cys-Met-Cly-Met-Thr-Ser-Arg-Tyr-Phe-Tyr-Asn-Gly-Tr-Ser-Met-Ala -Cys-Glu-Thr-Phe-Gln-Tyr-Gly-Gly-Cys-et-Gly-Asn-Gly-Asn-Asn-Phe-Val-Thr-Glu-Lys-G1u-Cys-Leu-G 1 n- Thr-Cy s- Arg-Thr- Va 1-Ala- Ala-Cys-Asn-Leu-Pro-I le-Val-Arg-Gly-Pro-Cys-Arg-Ala-Phe-l le-Gln -Leu-Trp-Ala-Phe- Asp-Ala-Vato Lys-Gly -shi ys-Cys-Vato Leu-Phe- Pro-Tyr-Cly-Gly-Cys-Gln-Gly-Asn-Gly-Asn- Ys-Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu-Tyr-Cys-Gly-Val-Pro-Gly-Asp-Cly-Asp-Glu- (Formula V)

G 1 u-Leu-shi eu-Arg-Phe

The amino acid sequence of nitz-type domain 1 specifically refers to the amino acid sequence represented by the following formula VI, and the amino acid sequence of K unitz-type domain 2 specifically corresponds to the following formula:アミノ酸 Refers to the amino acid sequence represented by Ι.

Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr- Ser-

Ala-Gly-Pro-Cys-llet-Gly-yet-Thr-Ser-Arg-

Tyr-Phe-Tyr-Asn-Gly-Tr-Ser-Met-Ala-Cys-

G 1 u-Thr-Phe-G 1 n-Tyr-C 1 y-G 1 y-Cys-Me t-Gly- (Formula VI)

Asn-Gly-Asn-Asn-Phe-Va 1 -Thr-Glu-Lys-Glu-

Cys-Leu-Gln-Thr-Cys-Arg Thr-Val-Ala-Ala-Cys-Asn-Leu-Pro-Ile-Val

Arg-Gly-Pro-Cys-Arg-Ala-Phe- Ile-Gln-Leu-

Trp-Ala-Phe-Asp-Ala-Val-Lys-Gly-Lys-Cys-

Val-Leu-Phe-Pro-Tyr-Gly-Gly-Cys-Gln-Gly-

Asn-G 1 y-Asn-Lys-Phe-Tyr-Ser-Glu-Lys-Glu- (Formula ΠI)

Cys-Arg-Glu-Tyr-Cys-Gly-Val-Pro-Gly-Asp-

Gly-Asp-Glu-Clu-Leu-Leu-Arg-Phe

The DNA encoding the amino acid sequence of UTI is defined as the Pichia yeast transformed by introducing the DNA into Pichia yeast by an appropriate method and transforming the Pichia yeast. Thus, any DNA having any base sequence may be used as long as it can express a protein having the amino acid sequence of UTI. The same applies to DNA encoding the amino acid sequence of Kunitz type domain 1 and DNA encoding the amino acid sequence of Kunitz type domain 2.

As the DNA encoding the amino acid sequence of UTI, preferably a DNA having a base sequence represented by base numbers 661-1095 among the base sequences shown in FIGS. 3 to 7 as a part of the base sequence. A.

The DNA encoding the amino acid sequence of Knitz-type domain 1 is preferably a nucleotide sequence represented by nucleotide numbers 724 to 891 in the nucleotide sequences shown in FIGS. 3 to 7 as a part of the nucleotide sequence. Is a DNA having

The DNA encoding the amino acid sequence of Knitz-type domain 2 is preferably represented by base numbers 892-2010 of the base sequences shown in FIGS. 3 to 7 as a part of the base sequence. It is a DNA having a base sequence.

In consideration of codon degeneracy, each of the DNAs according to the present invention may be one or more of the base sequences shown in FIGS. 3 to 7 as long as it can encode the amino acid sequence of UTI. May be replaced by another base. The origin and the like of the expression vector of the present invention are not particularly limited as long as it contains the above DNA and can express the DNA in Pichia yeast. In addition, the expression vector preferably has, in addition to the above DNA, a promoter, a terminator, a homologous region, a marker gene, and the like necessary for expressing the DNA in Pichio yeast. It is preferable that the vector has a base sequence encoding a signal peptide. In addition, it may carry an autonomous replication sequence or the like that can be replicated in a host. The base sequence encoding the promoter and signal peptide may be any sequence that functions in Pichia yeast.

Examples of the promoter include the A〇X1 promoter [Japanese Patent Laid-Open No. 63-39484 (EP-A-244584)], the DAS promoter, and the mutant AOX2 promoter. No. 1 (Japanese Unexamined Patent Publication No. Hei 6-99068 (EP-A-56040)) and the like can be used.

Examples of the signal peptide include yeast invertase (SUC 2) [Japanese Patent Application Laid-Open No. 60-41888 (EP-A-127304)], α-factor [Japanese Patent Application Laid-Open No. 1 1 もの 1 号酵母酵母酵母酵母酵母酵母酵母酵母酵母酵母酵母酵母酵母酵母酵母酵母 HS 酵母酵母酵母酵母, A シグナル HS HS HS HS No. 5 (EP—A—3 196 4 1)], an artificially created signal sequence [JP-A 1-240191 (EP—A—3 291)] Etc. can be used. As the evening light, AOX 1 evening light and evening [Japanese Patent Application Laid-Open No. 63-39584 (EP-A-24444598)] can be used. Examples of the homologous region include HIS4 (Japanese Patent Application Laid-Open No. 61-108391 (EP-A-1808999)), URA3, LEU2, and ARG4.

As the marker gene, an antibiotic resistance gene, an auxotrophic complement gene, or the like is used. Examples of antibiotics include cycloheximide, G-418, chloramphenicol, bleomycin, hygromycin and the like. The auxotrophic complement gene includes HIS 4, URA 3, LEU 2, ARG 4, and the like. The expression vector of the present invention is simply a plasmid vector or a plasmid vector having in advance a nucleotide sequence necessary for expression, production and secretion of UTI such as a promoter, a signal peptide-encoding nucleotide sequence or each Knitz-type domain thereof. A phage vector is prepared by introducing the above DNA into a first-class phage vector according to a conventional method (eg, Molecular Cloning, a laboratory manua 1, Second edition, edited by T. Maniatis, Cold Spring Harbor Laboratory, 1989). Can be. Alternatively, a nucleotide sequence required for expression can be chemically synthesized and introduced into a plasmid vector or the like together with the above DNA.

The present invention also relates to a Pichia yeast transformed with the above-described expression vector of the present invention.

The transformant was prepared by the spout plast method (Cregg T. et al., Mol. Cell. Biol. 5.3 pp. 376-3385, 1985), the alkali cation method Utoh H. et al., J. Bacteriol. 153, 163- 168), by transforming a yeast belonging to the genus Pichia with the vector of the present invention.

Examples of the Pichia yeast to be transformed include Pichia pastoris GTS115 strain (NRRL accession number: Y-15851).

When the transformant of the present invention secretes a protein having the amino acid sequence of UTI or its respective Kunit I type domain, the vector to be introduced into the transformant encodes those proteins. Those having a base sequence encoding the aforementioned signal peptide in addition to the above DNA are preferable.

The Pichia yeast, which is the transformant of the present invention, more preferably has the property of producing a protein having the amino acid sequence of UTI or each domain thereof and secreting the protein out of the transformant.

The present invention also provides a method for producing a protein having an amino acid sequence of at least one Knitz type domain of U UI by culturing the above-mentioned transformant (Pichia yeast), and transforming the protein from the culture. Collecting a protein having an amino acid sequence of at least one Knitz-type domain of UTI. W / 0 03

Related.

The manufacturing method is characterized by performing the following steps (a) to (c).

(a) Obtain a DNA having a nucleotide sequence encoding a protein having at least one kind of Kunitz type domain amino acid sequence of UTI.

(b) Pichia yeast is transformed with the expression vector containing the DNA obtained in (a) to obtain a transformant.

(c) culturing the transformant, and collecting a protein having at least one kind of Knitz type domain amino acid sequence of UTI from the resulting culture.

Here, a protein having an amino acid sequence of at least one kind of Kunitz-type domain of UTI refers to a protein having at least the amino acid sequence of either Kunitz-type domain 1 or Kunitz-type domain 2 of UTI. Means Therefore, if it has the amino acid sequence of Kunitz-type domain 1 or Kunitz-type domain 2, it may have another amino acid sequence at its N-terminal and / or C-terminal. A protein having an amino acid sequence is also included.

The Pichia yeast, which is a transformant, may be cultured according to a general method used for culturing Pichia yeast, for example, a method described in JP-A-6-22784.

Specifically, the medium may be a 0.01% to 8% methanol-containing YNB liquid medium [0.7% Yeast Nitrogen Base w / o Amino Acids (Difco)] and a 0.01% to 8% methanol-containing medium. YP medium [1% Yeast Extract (Difco), 2% Peptone (Difco)] and the like are exemplified.

The cultivation is usually carried out at 15 to 43 (preferably about 20 to 30 ° C.) for about 20 to 360 hours, and if necessary, aeration and stirring can be applied.

Subsequently, the target protein produced by the yeast of the genus Pichia is collected from the culture of the yeast of the genus Pichia. When the produced protein is not secreted outside the Pichia yeast, it is preferably isolated from the yeast cells of the genus Pichia, and when secreted, it is preferably isolated from the culture supernatant. The method of collecting the target protein from the culture can be performed by a method usually used for protein purification, for example, “Biochemical Experiment Lecture 1, Protein Chemistry” (edited by the Biochemical Society of Japan, 1979, The method can be carried out with reference to methods described in many documents such as Tokyo Chemical Dojin.

The obtained protein can be further purified if necessary. Purification methods include salting-out, ultrafiltration, isoelectric precipitation, gel filtration, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, chromatofocusing, and adsorption. Chromatography and reversed-phase chromatography Examples include various types of chromatography.

The present invention also relates to novel polypeptides having useful protease inhibitory activity.

The novel polypeptide of the present invention is characterized in that it has protease inhibitory activity. Specifically, at least one enzyme among eye enzymes such as tribsine and elastase, inflammation-related enzymes such as neutrophil elastase, closin and fibrinolytic enzymes such as plasmin, factor-1Xa, and kallikrein. It shows inhibitory activity. The novel polybeptide of the present invention preferably has a neutrophil elastase inhibitory activity that is stronger than the neutrophil elastase inhibitory activity of natural UTI derived from urine, and more preferably It also has an inhibitory activity on tribsine. The novel polybeptide of the present invention may be any amino acid sequence as long as it has the above-mentioned characteristics as long as it contains the amino acid sequence of the following formula I as at least a part of the amino acid sequence defining its primary structure. May also be used. Cys-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-Cys-Ile-Ala-Phe-Phe-Pro-Arg-Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-Met-Ala -Cys-Glu-Thr-Phe-Gln- Tyr-Gly-Gly-Cys-Met-Gly-Asn-Gly-Asn-Asn- (Formula I)

Phe-Val-Thr-Glu-Lys-Glu-Cys-Leu-Gln-Thr-Cys

That is, the polypeptide of the present invention may be a polypeptide defined by the amino acid sequence itself represented by Formula I, or an arbitrary amino acid sequence at the N-terminal and / or C-terminal of the amino acid sequence of Formula I. It may be a polypeptide to which two or more amino acids have been added.

For example, the novel polypeptide of the present invention may have an amino acid sequence represented by the following formula VII, formula II or formula VII on the N-terminal side thereof.

Lys-Glu-Asp-Ser (Formula VI I)

Ala-Yal-Leu-Pro-Gln-Glu-Glu-Clu-Gly-Ser-

Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr- (Formula II)

Lys

Ala-Val-Leu-Pro-Gln-Glu-Glu-Glu-Gly-Ser- Gly-Gly-Gly-Gln-Leu-Val -Thr-Glu-Val -Thr-Lys-Lys-Glu-Asp-Ser (Formula VI II)

In particular, a polypeptide having an amino acid sequence represented by the formula II on the N-terminal side is converted into a polypeptide having no amino acid sequence due to having the amino acid sequence. The expression level is remarkably large in comparison, and it is useful in that it can be produced in large quantities by recombinant DNA technology.

Further, the novel polypeptide of the present invention may have an amino acid sequence represented by the following formula II on the C-terminal side of the amino acid sequence.

Thr-Val-Ala-Ala-Cys-Asn-Leu-Pro-Ile-Val

Arg-Gly-Pro-Cys-Arg-Ala-Phe-I le-Gln-Leu-

Trp-Ala-Phe-Asp-Ala-Val-Lys-Gly-Lys-Cys-

Val-Leu-Phe-Pro-Tyr-Gly-Gly-Cys-Gln-Gly-

Asn-Gly-Asn-Lys-Phe-Tyr-Ser-Glu-Lys-Glu- (Formula Π Ι)

Cys-Arg-Glu-Tyr-Cys-Gly-Val-Pro-Gly-Asp-

Gly-Asp-Glu-Glu-Leu-Leu-Arg-Phe

Such a novel polybeptide is useful in that it has not only neutrophil elastase inhibitory activity but also tribcine inhibitory activity. Examples of such a polypeptide include a polypeptide represented by the following formula (IX).

Lys-Glu-Asp-Ser-Cys-GlD-Leu-Gly-Tyr-Ser-

Ala-Gly-Pro-Cys-I le-Ala-Phe-Phe-Pro-Arg-

Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-Met-Ala-Cys-

Glu-Thr-Phe-Gln-Tyr-Gly-Gly-Cys-Met-Gly-

Asn-G 1 y-Asn-Asn-Phe-Va 1 -Thr-G 1 u-Lys-G 1 u-

Cys-Leu-Gln-Thr-Cys-Arg-Thr-Val-Ala-Ala-

Cys-Asn-Leu-Pro- 11 e-Val-Arg-Gly-Pro-Cys-

Arg-A 1 a-Phe- I le-Gl n-Leu-Trp-Ala-Phe- Asp-

Ala-Val-Lys-Gly-Lys-Cys-Val-Leu-Phe-Pro-

Tyr-Gly-Gly-Cys-Gln-Cly-Asn-Gly-Asn-Lys-

P e-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu-Tyr- (Formula IX)

Cys-Gly-Val-Pro-Gly-Asp-Gly-Asp-Glu-Glu-

Leu-Eu-Arg-Phe Another preferred example of the novel polypeptide of the present invention is a polypeptide having an amino acid sequence represented by the following formula IV.

Ala-Va 1 -Leu-Pro-G in-Glu-Glu-Glu-G 1 -Ser-

Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr-

Lys-Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr-

Ser-Ala-Gly-Pro-Cys-I le-Ala-Phe-Phe-Pro-

Arg-Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-Met-Ala-

Cys-G 1 u-Thr-Phe-Gln-Tyr-Gly-Gly-Cys-Me t-

Gly-Asn-Gly-Asn-Asn-Phe-Val-Thr-Glu-Lys-

G 1 u-Cys-Leu-G 1 n-Thr-Cys-Arg-Thr-Va 1-Ala-

Ala-Cys-Asn-Leu-Pro-I le-Val-Arg-Gly-Pro-

Cys-Arg-Ala-Phe-I le-Gln-Leu-Trp-Ala-Phe-

Asp-Ala-Val-Lys-Gly-Lys-Cys-Val-Leu-Phe-

Pro-Tyr-Gly-Gly-Cys-Gln-Gly-Asn-Gly-Asn-

Lys-Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu- (Formula IV)

Tyr-Cys-Gly-Val-Pro-Gly-Asp-Gly-Asp-Giu-

GIu—Sheu—Sheu—Arg—Phe

The polypeptide of the present invention includes not only the amino acid sequence represented by Formula IV or Formula IX but also the polypeptide defined by itself, and the N-terminal and Z or C of the amino acid sequence represented by Formula [V or Formula. It also includes a polypeptide having one or more optional amino acids added to the terminal. More preferably, it is a peptide having an amino acid sequence represented by Formula IV or Formula IX, and having a trypsin inhibitory activity and a neutrophil elastase inhibitory activity, wherein the neutrophil elastase inhibitory activity is a natural UTI derived from urine. It is significantly enhanced as compared to the activity.

1 x iO following specifically, inhibitory activity K i value for neutrophils Heras evening over Ze, more preferably K i values in 5 X 10 - of the ^{invention 1 0} M or less is polypeptides are exemplified Novel polypeptides have extremely strong neutrophil elastase inhibitory activity And can be produced in large quantities using recombinant DNA technology. In particular, polypeptide having an amino acid sequence represented by the formula II on the N-terminal side is useful in that the expression level is extremely high.

In addition, the novel polypeptide of the present invention is expected to have an amino acid sequence partially common to UTI, to be high in tissue permeability due to being a small molecule, and not to exhibit antigenicity to humans. You. Further, it is useful as a material for producing a fusion protein, for example, by fusing another polypeptide having another function to the N-terminus and / or the C-terminus as necessary.

The novel polypeptide of the present invention may or may not have a sugar chain. The origin of the acquisition is not particularly limited. For example, it may be chemically synthesized using a peptide synthesizer or the like. Preferably, it is a polypeptide produced using recombinant DNA technology. More preferably, it is a polypeptide produced by recombinant DNA technology using yeast or Pichia yeast or Escherichia coli as a host. In addition, the polypeptide may be chemically modified (for example, sugar addition, alkylation, etc.) as long as the specific activity is not substantially reduced. Further, a substance obtained by forming a complex with a pharmacologically acceptable acid or base or a polymer such as polyethylene glycol is also included in the form of the novel polypeptide of the present invention.

The present invention also relates to DNA having a nucleotide sequence encoding the novel polypeptide. The DNA may be an amino acid sequence represented by any one of Formula I, Formula IV or Formula IX, or other amino acids or peptides at the N-terminal and Z- or C-terminals of the amino acid sequence thereof (for example, Formula I I. VI I, VI II or those having the amino acid sequence represented by Formula III), or any other nucleotide sequence within a range that does not cause a change in the amino acid sequence of the polypeptide. Good.

Specifically, as the DNA encoding the polypeptide represented by the formula I, of the nucleotide sequences shown in FIGS. 3 to 7, the nucleotide sequence represented by nucleotide numbers 736 to 888 from the 5 ′ side The base number of 766 to 780 is ATCGCTTTCT TTCCT

And a DNA having a nucleotide sequence substituted by As the DNA encoding the polypeptide represented by the formula [X], the nucleotide sequence of the nucleotide sequence represented by nucleotide numbers 724 to 1095 from the 5 'side of the nucleotide sequence shown in FIGS. 766 to 780 bases are

ATCGCTTTCT TTCCT

And a DNA having a nucleotide sequence substituted by In addition, as the DNA encoding the polypeptide represented by the formula IV, in the base sequences shown in FIGS. 3 to 7, bases represented by base numbers 661 to 1095 from the 5 ′ side are used. Nucleotide number 766-780 of the sequence is

ATCGCTTTCT TTCCT

And a DNA having a nucleotide sequence substituted by

Further, as the DNA encoding the polypeptide represented by Formula II, a DNA having a base sequence represented by base numbers 61 to 723 from the 5 'side of the base sequences shown in FIGS. 3 to 7, As the DNA encoding the polypeptide represented by the formula Vil, a DNA having a base sequence represented by base numbers 724 to 35 from the 5 'side of the base sequence shown in FIGS. Examples of the DNA encoding the polypeptide shown in [1] include, among the nucleotide sequences shown in FIGS. 3 to 7, a DNA having a nucleotide sequence represented by base numbers 661 to 735 from the 5 ′ side. The DNA encoding the polypeptide represented by the formula III is, for example, a base sequence represented by base numbers 892-2010 from the 5 'side of the base sequences shown in FIGS. 3 to 7. DNA having the following is exemplified.

In consideration of the fact that a plurality of codons correspond to one amino acid, the DNA of the present invention has, in the base sequence specified in FIGS. Also included are sequences having a base sequence in which one or more bases have been replaced with other bases.

In addition, the DNA of the present invention has a 5′-terminal in addition to the nucleotide sequence specified in the above figure. And one or more arbitrary bases added to the 3 ′ end. The base to be added may be any base or base sequence as long as it does not cause a change in the amino acid sequence encoded by the base sequence to be added by being added to the above base sequence. Good.

As an example, the base sequence added to the 5 ′ end includes the base sequence ATG as a start codon, and the base sequence added to the 3 ′ end includes the stop codon TAA, TAG or TGA and the like.

The DNA of the present invention is a nucleotide sequence encoding another amino acid or polypeptide at the 5 ′ end and Z or 3 ′ end of the nucleotide sequence encoding the amino acid sequence of the novel polypeptide of the present invention. May be provided.

The DNA of the present invention may be obtained by any method. For example, a method of chemically synthesizing with reference to the nucleotide sequences shown in FIGS. 3 to 7, a method using recombinant DNA, using an appropriate chromosomal DNA library or cDNA library as a material, etc. There is a way to get it.

The chemical synthesis of the DNA of the present invention is performed, for example, as follows. A desired DNA having a nucleotide sequence encoding the novel polypeptide of the present invention is designed, and if necessary, the designed DNA is divided into appropriate fragments, and an oligomer corresponding to each fragment is converted into a fully automatic DNA synthesizer. (Eg, Type 381 A, manufactured by Applied Biosystems).

On the other hand, as a method for obtaining the DNA of the present invention using recombinant DNA technology, an appropriate cDNA library, a chromosomal DNA library, or an amino acid sequence represented by Formula I or Formula IV is encoded. Site-directed mutagenesis using DNA or the like as material (hereinafter referred to as Site-directed mutagenesis, Kramer. W, et al., Ucleic Acid Res., 12, 944, 9456, 1984 and Kunkel, TA, etc. Enzymology, Vol. 154, pp. 367-382, 1987), and a method of modifying a base sequence and amplifying DNA by a known method such as PCR.

Further, the present invention provides a DNA having a nucleotide sequence encoding the novel polypeptide described above. 9

The present invention relates to a vector characterized by containing

The origin of the vector is not limited as long as it contains DNA having a nucleotide sequence encoding the novel polypeptide of the present invention. For example, the DNA of the present invention was introduced into an appropriate position in various plasmid vectors such as pBR322, pUC18 and pUC19, and phage vectors such as λgt10 and λgt11. Things. Preferably, it is a vector that can be replicated in the host cell to be used or a vector that can be integrated into the host chromosome. More preferably, it is a vector that can be replicated in Escherichia coli or yeast, preferably yeast of the genus Pichia, or that can be integrated therein. K It is the evening.

The vector of the present invention comprises, in addition to DNA encoding the novel polypeptide of the present invention, bases such as a promoter and an optional ribosome binding site necessary for expressing the novel polypeptide of the present invention in a host. It is preferable to have a sequence. Particularly, when the vector of the present invention is a vector for secretion expression, a vector further having a base sequence encoding a signal peptide and the like is preferable. The above-mentioned promoter, the ribosome binding site, the nucleotide sequence encoding the signal peptide, and the like may be any sequence that functions in the host used. Examples of the promoter and signal peptide include those described above.

The vector of the present invention simply has in advance a nucleotide sequence necessary for the expression, production and secretion of the novel polypeptide of the present invention, such as a promoter, a ribosome binding site, and a nucleotide sequence encoding a signal peptide. The DNA of the present invention is introduced into a brasmid vector or a phage vector according to a conventional method (eg, Molecular Cloning, a laboratory manual, Second edition T. Maniatis, etc., Cold Spring Harbor Laboratory, 1989). It can be manufactured by the following. Alternatively, it is also possible to chemically synthesize a nucleotide sequence required for expression and introduce it into a plasmid vector or a phage vector together with the DNA of the present invention.

The present invention also relates to a transformant transformed with a vector comprising a DNA having a nucleotide sequence encoding the novel polypeptide. The transformant was prepared by the calcium chloride method, the rubidium chloride method, Hanahan. D, Techniques for Transformation of E. coli. In: DNA cloning vol 1, Gl over, DM (ed.) 109-136, IRL press, 1985) and by transforming a suitable host cell with the vector of the present invention.

When yeast is used as a host cell, the blast method (Hinnen A. et al., Proc. Natl. Acad. Sci. USA 75, 1929-1933, 1978, Cregg JT et al., OLCell. Bio 1.5, p. 3376-3385 198) and the known method such as the alkali cation method (Itoh H. et al., J. Bacteriol. 153, pp. 163-168, 1983).

The transformant of the present invention more preferably has a property of producing the novel polypeptide of the present invention and secreting it outside the transformant.

When the transformant of the present invention secretes the novel polypeptide of the present invention out of the transformant, the introduced vector may be a signal peptide in addition to the DNA of the present invention. Those having an encoding base sequence are preferred.

As the host cell to be used, any eukaryotic cell typified by yeast or the like can be used as long as it is suitable for expression and production of the polypeptide of the present invention. It may be a biological cell. Preferably, Escherichia coli and yeast are used, and more preferably, Pichia yeast is used.

Further, the present invention provides a novel polypeptide of the present invention, which comprises culturing the above-mentioned transformant, producing the novel polypeptide of the present invention, and collecting the polypeptide from the obtained culture. It is a manufacturing method.

The production method is a method for producing a novel polypeptide of the present invention, which comprises performing the following steps (a) to (c).

(a) A DNA having a nucleotide sequence encoding the novel polypeptide of the present invention is obtained.

(b) Transform a host cell with the expression vector containing the DNA obtained in (a) to obtain a transformant.

(c) culturing the transformant, and collecting the novel polypeptide of the present invention from the resulting culture. Culture of the transformant is performed by a general method used for culturing microorganisms or animal cells, that is, “Biochemical Engineering” (written by Shuichi Aiba et al., 1976, University of Tokyo Press) or “Tissue Culture”. (Junnosuke Nakai et al., 1977, Asakura Shoten), etc.

Subsequently, the novel voriveptide of the present invention produced by the transformant is collected from the culture of the transformant. When the produced polypeptide is not secreted outside the transformant, it is preferably isolated from the transformant, and when secreted, it is preferably isolated from the culture supernatant. The collection and purification of the polypeptide can be carried out according to a conventional method as described above.

The present invention also relates to a method for enhancing expression of a polypeptide having a protease inhibitory activity in a transformant in a large amount. As described above, the present inventors have found that a polypeptide having an amino acid sequence represented by the formula II on the N-terminal side is more effective than a polypeptide having no amino acid sequence represented by the formula に on the N-terminal side. It was found that the expression level in yeast was remarkably large. Therefore, by binding the amino acid sequence represented by the formula H to the N-terminal side of olibeptide having a protease inhibitory activity such as neutrophil elastase and trypsin, the polypeptide is added to the N-terminal side. By expressing the amino acid sequence represented by the formula II with the amino acid sequence added thereto, a large amount of a polypeptide useful as a protease can be obtained. That is, a vector is prepared in which a nucleotide sequence encoding an amino acid sequence represented by the formula I is linked to the 5 'end of a nucleotide sequence encoding an amino acid sequence containing a protease inhibitory activity site, and this vector is used. As a result, the expression of borobeptide useful as a protease inhibitor in a transformant is enhanced, and a large amount of protease inhibitor can be obtained. One or more amino acids may be arranged between the amino acid sequence of the protease inhibitory active site and the amino acid sequence of the formula II.

The polypeptide to which the amino acid sequence represented by the formula 11 can be added to the N-terminus at the N-terminus is not limited to the novel polypeptide of the present invention having the amino acid sequence represented by the formula I or IX described above. , Neutrophil elastase, trypsin and other proteases Since the method can be applied to other polypeptides having an inhibitory activity, the expression enhancing method of the present invention is extremely useful.

According to the present invention, it is possible to provide a method for producing UTI and its respective K unitz-type domains by a genetic engineering technique using yeast of the genus Pichia as a host cell, and an expression system related thereto. . An expression vector comprising a DNA encoding the UTI of the present invention or a Kunitz-type domain thereof, an expression system comprising Pichia yeast transformed with the vector, and a UTI using the same and each of them. The method for producing a K unitz-type domain enables simple and efficient mass production of UTI and its respective K unitz-type domain. In addition, by using this system, mutants or derivatives of UTI and its respective K unitz-type domains can be easily produced, so that new protease inhibitors, such as the development of useful protease inhibitors by studying structure-activity relationships, can be used. It can contribute to drug development.

Further, the present invention provides a novel and useful polypeptide, which has one or more protease inhibitory activities and is superior in neutrophil elastase inhibitory activity to urine-derived UTI, and is preferably a superior neutrophil elastase. It is possible to provide a useful novel polypeptide having an inhibitory activity and having a high expression level in a genetic engineering technique and capable of being produced in large quantities, and a method for producing the polypeptide by the genetic engineering technique.

According to the method for enhancing the expression of a protease inhibitor of the present invention, it is possible to remarkably increase the expression amount of a polypeptide having protease inhibitory activity in yeast. The method is extremely useful in that it can be applied to polybeptides having a protease inhibitory activity other than the novel polypeptide of the present invention.

Hereinafter, the present invention will be described with reference to Examples to further clarify the present invention. However, these are one aspect of the present invention, and the present invention is not limited by these. Various operations in the examples described below were performed with reference to the following magazines and books.

1. Laboratory Manual Genetic Engineering, Masami Muramatsu, 1989, Maruzen Co., Ltd.-

2. Genetic manipulation experiment method, edited by Yasutaka Takagi, 1980, Kodansha

3. Gene Manipulation Manual, edited by Yasutaka Takagi, 1982, Kodansha

4. Molecular Cloning, a laboratory manuaK Second edition, T. Maniatis, etc., 1989, Cold Spring Herba. Laboratory

5. Methods in Enzymoiogy, 65, L. Grossman et al., 1980, Academic Press

6. Methods in Enzymoiogy, Vol. 68, edited by R. Wu, 1979, Academic Press Example 1 Expression of UTI in Pichia yeast

(1) Cloning of UTI cDNA

① Preparation of probe

In the nucleotide sequence of UTI cDNA reported by JF Kaumeyer et al. (Nucl. Acids Res. 14 (20) .p7839-7850.1986), we focused on the region encoding the two inhibitor domains. Probes 1 and 2 were designed to have a GC content of 50% or more. The area of each probe is shown below.

796 837 • Probe 1 GGTACATCCA TGGCCTGTGA GACTTTCCAG TACGGCGGCT GC

1048 1089 • Probe 2 GAGTACTGCG GTGTCCCTGG TGATGGTGAT GAGGAGCTGC TG

Oligonucleotides were synthesized on a DNA synthesizer (Model 392, manufactured by Applied Biosystems Japan) using a 0.2 M FOD column (manufactured by Ablied Biosystems Japan) based on this sequence.

The synthesized oligonucleotides are used in a 0PC cartridge After purification using Thames, Japan, the gel was electrophoresed with a 7M urea 10% acrylamide-modified gel. As a result, both probes showed a single band.

These oligonucleotides 7 - ³ was end labeled with P ATP. Label 7 oligonucleotide and 4.6MBq of Loong - mixing the ³² P ATP (1 molar ratio: 5), Ri by the reacted according to a conventional method using T4 p oly nucleotide kinase (Takara Shuzo) went.

② Screening

(a) —Next Screening

Human liver-derived c DNA λ gt 1 0 library one (Kurontetsuku Co.) SM buffer to the phages liquid becomes 4.2 xiO ⁵ pfu / ml of _{(lOOmM NaCl, 10mM MgS0 4,} 50mM Tris-HCl (pH7 .5), diluted with 0.001% gelatin), was overlaid after mixing with the 100 \ and diluted indication bacterial solution, the mixture TB10 bottom agar plate (1.5% ager / ΤΒΙΟ medium, the phi 15cm), 37 ^e Incubated with C for about 7 hours.

The dilution indicator bacterial solution was prepared as follows.

E. c01iC600hf1 strain was inoculated into a 5 ml 1L medium, and cultured at 37'C for 9 hours. This was inoculated in its entirety into 500 ml of TBIO medium dispensed into a 2 L kolben, and cultured at 37 ° C. The turbidity at 60 0 nm of the culture was bacteria collection after was measured, after washing the obtained cells with 1 0 mM MgSO ₄ solution, 25 m l 01 Omm MgSO, suspended in an aqueous solution (turbidity OD _e .. = 3.1.2), then OD _{eo in} 1 OmM Mg SC aqueous solution. = 2 and used.

The culture was stopped when the blacks became visible, and stored at 4 ° C as a master plate. Two membranes (colony / plaque screen, manufactured by DuPont) were prepared per master plate. The resulting membrane was incubated on a TB10 bottom agar plate at 37 ° C- 晚 to amplify phage. These membranes were immersed in a 0.5N aqueous solution of sodium hydroxide for 5 minutes, and subjected to a denaturation treatment. After repeating the same operation, bacterial cell residues were removed with paper. Then, the plate was immersed in 1M Tris-HCl buffer (PH7.5) for neutralization, and rinsed with 2 × SSC buffer. Put these on filter paper After air-drying, the cells were incubated in a prehybridization solution (2 × SSC.2% SDS, 5 × Denhardt's soln., 500 Aig / ml yeast tRNA) at 60 ° C. for 4 hours.

After pre-hybridization, transfer the membrane to a new bag, and use a hybridization solution for each probe (2 X SSC, 2% SDS, 5x Denhardt's soln., 100 l / g / rnl yeast tRNA, after the solution added to heating and quenching treatment was intended) containing each probe end-labeled, were hybrida I See Chillon 40 ^e C-evening.

Then, the membrane was washed with 2 x SSC-2¾ SDS aqueous solution for 20 minutes at room temperature, 2xSSC-2% SDS aqueous solution at 52 ° C for 20 minutes, and further washed with 1 XSSC-2% SDS aqueous solution at 45 ° C for 40 minutes. (Registered trademark) and exposed to light at 180 ° C.

As a result of confirming the signals, each membrane had 30 to 40 specific signals, and a total of about 400 signals were obtained. Most signals were consistent for probes 1 and 2.

(b) Secondary screening

— From the master plate used for the next screening, remove the top agarose of 10 spots whose signal has been matched with the probe 1 and 2 from the master plate, pulverize in 0.5 ml SM buffer and finely crush. Phage was eluted by adding 1 drop of mouth form and leaving at 2.5 for 2.5 hours. The concentration of the phage eluate assuming 1 xiO ⁷ pfu / ml, was Mare积three stages of ^{^{5 X 10 3, 1 10 3}} . 5 xiO 2 pfu / ml in SM buffer. These files - mixing the di-dilution 100 fi \ and diluted indicator strain was 100 ^ 1, and Ichiboku base 37 Incubate ^e C20 minutes. Add 3ml TB10 top agarose and layer on a TB10 botto magagar plate (09cm) pre-warmed at 37 ° C. C-晚was incubated, 5 xiO ² pfu / 10 in dilution system set ml ³ - ⁴ pfu / ml of the plaque was observed. Therefore, in the next membrane preparation, a system mainly set to 5 xiO ² pfu / ml was used.

-As in the next screening, the phage was not amplified on the membrane at 37 ° C for 6 hours and stored at 4. The membrane was alkali-denatured, neutralized, and rinsed with 2 x SSC, and then incubated in a prehybridization solution (2XSS 2% SDS. 5x Denhardfs soln., 100 / g / ml yeast tRNA) for 60'C for 6 hours. . After prehybridization, hybridization was performed using probe 2 in the same manner as in primary screening. Each membrane was washed twice with 1 × SSC-2 SDS aqueous solution at room temperature for 10 minutes. Thereafter, each membrane was covered with Saran Wrap (registered trademark) and exposed at room temperature for 6 hours.

As a result of developing this, the bottling in which a signal was observed was subjected to tertiary screening.

(c) Tertiary screening

The plaque corresponding to the signal from each master plate used for secondary screening was mixed with a toothpick using indicator bacterial plate (100/1 diluted indicator bacterial solution and 3 ml TB10 top agarose were mixed at 37 ° C in advance. Inoculated on a warmed TB10 bot tom agar plate). This was incubated at 37 ^e C— 晚.

Of amplification were performed 37'C3 hours phage on the membrane, alkaline denaturation, After performing neutralization treatment, and 2 x SSC Li Nsu, while handling blur hybrida I See Chillon solution 2 6 0 ^e C. Incubated for 5 hours.

After blur hybridization, probe 1 was used for hybridization in the same manner as in primary screening.

After washing each membrane with a 1 × SSC-2% SDS aqueous solution at room temperature for 10 minutes, the membrane was covered with Saran Wrap (registered trademark) and exposed at room temperature for 6 hours using a sensitizing screen.

As a result of development, 1 to 5 strains of phage clones considered to be purified were obtained for each spot.

(d) Subcloning

Phage DNA was prepared from 16 of the obtained phage clones, and the obtained phage DNA was digested with restriction enzyme EcoRI, followed by agarose gel electrophoresis to confirm the insert. Most phage clones had one or two inserts with a size of 0.5-2 kb. Furthermore, Southern hybridization using probe 2 was performed, and a 1.2-1.3 kb fragment reported in the literature (JF Kaumeyer et al. Nucl. Acids Res. 14 (20), p 7839-7850, 1986) was reported. 6 claw with insert CDNA was excised from the DNA and subcloned into the EcoRI site of pUC19. Restriction enzyme mapping was performed, and the nucleotide sequence was confirmed using the obtained plasmid DNA.

(e) Confirmation of nucleotide sequence

The nucleotide sequence was confirmed by the dideoxy method using [a-32p] dCTP. Specifically, Sequenase ver.2 (Pharmacia) and 7-DEAZA Sequenceing kit ver.2 (Takara Shuzo) were used according to the protocol.

As a result, the nucleotide sequence of the obtained UTicDNA was exactly the same as that reported by Kaumyer et al. (JF Kaumeyer et al., Nucl. Acids Res. 14 (20), p7844-7845, 1986) (FIG. 3). ~ Figure 7).

Hereinafter, the operation was performed using the No. 7 strain of which the entire nucleotide sequence of UT I cDNA was confirmed among the obtained subcloning strains.

③ UT I cDNA cassette

Examination of the nucleotide sequence of the UTI cDNA obtained in Example 1 (1) revealed that substituting G at nucleotide position 660 with C creates a new restriction enzyme Aoro51HI site (base number is According to a report by Kamnyer et al.). The generation of the Aoro51HI site could cleave the gene at the site corresponding to the N-terminus of the native UTII, and was considered to be extremely effective in the future construction of expression systems. The UTI structural gene consists of 441 bases and encodes 147 amino acids. On the other hand, the C-terminal amino acid of natural UTI is mainly Leul43, and the existence of a sequence having Argl44 or Phel45 has also been reported (Hochstrasse K. et al. Hoppe-Seyler's Z. Phisiol. Chem. 365, pll23_1230, 1984), and no sequence having Asnl47 at the C-terminus has been reported. Therefore, when UTI is expressed by genetic engineering, it is considered preferable to use a sequence found in nature, so Phel45 was used as the C-terminal amino acid. In consideration of the above points, UTIcDNA was cassetted.

Synthesized DNA (Fig. 8) of the 5'-end and 3'-end regions of the UTI gene corresponding to the above modification was converted to a DNA synthesizer (Applied Biosystems Japan, model 392). The synthetic DNA was designed so that both ends become the cohesive ends of each restriction enzyme after annealing. In addition, the synthetic DNA corresponding to the C-terminal coding region has a sequence in which 6 bases corresponding to 2-amino acid at the C-terminal have been deleted and 21 bases in the 3 'untranslated region have been deleted from an operational point of view. I made it.

On the other hand, DNA containing UTIcDNA obtained by digestion with Bbd at base number position 504 of UT Ic DNA contained in plasmid Ji7 of subcloning strain No. 7 to restore blunt ends, and further digesting with EcoRl. The fragment was subcloned into the Smal-EcoRI region of pUC19 (Π / Bbe ERI, Fig. 9). Based on this / Bb-ERI, each synthetic DNA was incorporated.

To replace the N-terminal coding region (hereinafter referred to as the N-terminal coding region), # 7 / BbeI-ERI was digested with BamHI and EcoRl, and a DNA fragment containing UTicDNA was recovered. This was digested with Sau3AI at the base number 686 of UT I cDNA to recover a Sau3A EcoRI fragment. In addition, # 7 / BbeI-ER [was digested with Aval and EcoRl at the base number 645 of UTI cDNA to recover the vector portion. The obtained DNA fragments and the synthesized DNA were ligated to obtain a clone pUTI-N in which the N-terminal coding region was modified (FIG. 10).

In order to replace the C-terminal coding region (hereinafter referred to as the C-terminal coding region), Π / BbeI-ERI was digested with Pst [and Ecol? The DNA fragment containing the latter half of the DNA was recovered. This was digested with Hhal at base number 1089 of UT I cDNA to recover a Pstl-Hhal fragment. In addition, # 7 / Bbeto ER I was digested with Pstl and Smal at base number 1146 of UTI cDNA to recover the vector portion. The obtained both DNA fragments and the synthetic DNA were ligated to obtain a clone ρΙΓΠ-C in which the C-terminal coding region was modified (FIG. 11).

Next, pUTI-N was digested with Pstl to recover a DNA fragment between the PsU site derived from PUC19 and the Pstl site at the base number 877 of the UTI cDNA. This was ligated to the Pstl site of pUTI-C to create a clone pUTIN-C in which the N-terminal and C-terminal coding regions were modified (FIG. 12). This plasmid was digested with Aor51HI-SmaI to obtain 3 ' UTI cDNA from which the poly A sequence of the translation region has been removed can be obtained.

(2) Creation of expression plasmid pHH310 (Fig. 14)

PA0807N [Japanese Unexamined Patent Publication No. 2-104290 (EP-A-0344459)] which is an expression plasmid of yeast belonging to the genus Pichia has an EcoRI site in the closing site. To cope with this, plasmid pUTIN-C carrying cassette UTI cDNA is digested with SmaI and treated with CIP (calf intestine alkaline phosphatase: Takara Shuzo Co., Ltd.). (Takara Shuzo Co., Ltd.), and the resulting cells were transformed into E. coli HB101 by a conventional method to produce plasmid PUT I NC / ER I (FIG. 13). By digesting this plasmid with Aor51HI and EcoRI, UTIcDNA from which the poly A sequence of the 3 'untranslated region has been removed can be obtained.

After the DNA corresponding to the yeast SUC2 signal peptide prepared for the construction of the UTI expression system (Chang CN, et al., Mol. Cell. Biol 6, pl812-1819. 1986), the T4 It was phosphorylated using kinase. This DNA is designed to have its 5 'end at the EcoRI junction end and its 3' end at the blunt end, and some base sequences have been modified according to the codon usage of Pichia yeast. (See Figure 15). Next, pUTIN-CNORI was digested with Aor51HI and EcoRI, and an approximately 0.4 kb DNA fragment containing the UTI gene was recovered. Incidentally, this DNA fragment has the 5 'end corresponding to the N-terminal of native UTI, and can be directly ligated to the SUC2 signal. In addition, in addition to the modification shown in FIG. 8, this DNA fragment has the Bori A sequence in the 3 ′ untranslated region removed.

Above, a synthetic SUC2 signal gene, pUTIN-C / ERI, prepared to have an EcoRI junction site at the 5 'end, was obtained by digestion with A0r51HI and EcoRI. After mixing and ligating the obtained DNA fragment of about 0.4 kb and the three DNA fragments of EcoRI digested pAO807N, the competent cell E.c01iHB1 01 was transformed. Plasmid DNA was prepared from the resulting transformant by a conventional method, treated with restriction enzymes and treated with AOX1 promoter and SUC2. The nucleotide sequence of the signal • UTI gene connecting region and the UTI gene • ΑΟΧ1 terminator one connecting region was confirmed, and the plasmid, which was an expression vector for Pichia yeast, was named ρΗΗ310. Figure 14).

(3) Preparation of expression strain and its properties

After PHH310 was linearized with the Stul site, a restriction enzyme on the selection marker HIS4, the Pichia pastoris GTS1 strain was targeted to the his4 locus on its chromosome by the spheroplast method (Cregg JT, Mol. Cell. Biol. 5. p3376-3385. 1985).

The obtained transformant was inoculated into 5 ml of YNB medium (0.7% yeast nitrogen base / o Amino Acids, 2% dextrose) and cultured at 30 ° C for 2 days. This was inoculated 10 times into 5 ml 2 x YP-2¾ glycerol medium (2% yeast extract, 4ept eptone, 2% glycerol), and cultured at 30 ° C for 2 days to grow the cells. This was centrifuged (2000 rpm, 5 min. At room temperature), and the obtained cells were treated with 5 ml of YP-2% methanol medium (1% Yeast Extract, 2% Peptone,

The cells were suspended in 2% methanol) and further cultured at 30 days for 5 days.

Centrifugation of the culture solution (lOOOOrpm, 5 minutes, 4 ^e C), the culture supernatant was collected. For the obtained culture supernatant, the activity of inhibiting tribsine and the activity of inhibiting human neutrophil elastase were measured according to the methods described in Reference Examples 2 and 3. As a result, the supernatant showed remarkable trypsin-inhibiting activity and a little human neutrophil elastase-inhibiting activity. Example 2 Construction of expression systems for nitz-type domain 1 and K unitz-type domain 2

An expression plasmid was constructed for expressing each of the two Kunitz-type domains (FIG. 2) constituting UTI in Pichia yeast. In the present embodiment, the Knitz-type domain 1 is a polypeptide obtained by adding the N-terminal peptide of UTI consisting of 21 amino acids to the N-terminus of the original Knitz-type domain 1, that is, A It was expressed as polybeptide consisting of 1a1 to Arg77 (hereinafter referred to as N-terminal peptide-added Knitz type domain 1).

Each Knitz-type domain expression plasmid was prepared as follows. (1) Construction of expression plasmid

① Preparation of synthetic DNA

In order to construct expression plasmids for each of the Knitz-type domain 1 and the Kunitz-type domain 2 with an N-terminal peptide, first, a synthetic DNA that complements the C-terminal coding region of the Kuniΐ-type domain 1 with an N-terminal peptide ( Synthetic DNA (Fig. 17) was prepared to complement the N-terminal coding region of Fig. 16) and Knitz-type domain 2. N-terminal peptide added Kunitz-type domain 1 C-terminal coding region synthesis DNA has a structure with a Pst I junction at 5 ', an EcoR I junction at 3', and a stop codon in the middle. . The synthetic DNA of the N-terminal coding region of Kunitz type domain 2 had a structure having a blunt end at the 5 ′ and an Ap I junction end at the 3 ′ to be linked to the SUC2 signal. In addition, since the restriction enzyme SphI site was created in the sequence without any amino acid change due to the single base substitution, C in base number 902 (see FIG. 6) of the UTI cDNA was changed to A. This newly provided SphI site is effective for re-linking Kunitz-type domain 1 and Kunitz-type domain 2 and modifying the N-terminal code region of Knitz-type domain 2 .

These syntheses were performed using a DNA synthesizer (Applied Biosystems Japan, model 392) based on the sequences shown in FIGS. 16 and 17.

(2) Preparation of expression vector for N-terminal peptide-added Kunitz-type domain 1 (N-terminal peptide-added Kunitz-type domain 1: base numbers 66 1 to 891 of UTI cDNA; see FIGS. 3 to 7) )

The pHH310 constructed in Example 1 (2) was digested with restriction enzymes EcoRI and PstI, and a fragment containing (SUC2 signal + Knitz type domain 1 structural gene with N-terminal peptide added) was cut out. . This fragment was ligated to the EcoRI site of pUC19 together with the synthetic DNA corresponding to the C-terminal coding region of Kunitz-type domain 1 shown in FIG. A plasmid pHH305 carrying the nitz-type domain 1 gene unit (see Fig. 18) was obtained (Fig. 19 ). The gene unit obtained by digesting this pHH305 with EcoRI was inserted into the EcoRI site of pA〇807N to create an N-terminal peptide-added Kunitz-type domain 1 expression plasmid pHH313 (Fig. 20).

(3) Preparation of expression vector for Kunitz type domain 2 (Kunitz type domain 2: salt of UT I cDNA ¾ numbers 892 to 1095, see FIGS. 3 to 7)

The pUTIN-CZERI constructed in Example 1 (2) was digested with restriction enzymes ApaI and EcoRI to obtain a DNA fragment of the Kunitz type 2 domain gene. This fragment and the synthetic DNA and synthetic SUC2 signal gene corresponding to the N-terminal coding region of Knitz-type domain 2 shown in Fig. 10 were ligated to the EcoRI site of pUC19, and secreted Kunitz A plasmid pHH306 carrying the type domain 2 gene unit (see FIG. 18) was obtained (FIG. 21). The gene unit obtained by digesting this pHH306 with EcoRI was inserted into the EcoRI site of PAO807N to prepare a Kunitz type domain 2 expression plasmid pHH314 (FIG. 22).

(2) Preparation of expression strain and its properties

After linearizing the expression brassid pHN3 13 of Kunitz-type domain 2 and the expression plasmid of Kunitz-type domain 1 with N-terminal peptide added in step (1) at St SI site In the same manner as in Example 1, Pichia yeast strain GTS115 was transformed.

The obtained transformant was inoculated into 5 ml of YNB medium and cultured at 30 ° C. for 2 days. This was inoculated in a 5 ml 2 YP-2P glycerol medium at 10%, cultured at 30 ° C. for 1 day, added with methanol to a final concentration of 2%, and further cultured at 30 at 3 days.

The culture solution was centrifuged (100 rpm, 5 minutes, 4 times), and the obtained culture supernatant was examined for its inhibitory activity against tribsine and human neutrophil elastase according to the methods described in Reference Examples 2 and 3. As a result, no culture supernatant of the N-terminal peptide-added Knitz type domain 1 expression strain was inhibited. On the other hand, the culture supernatant of the Kunitz-type domain 2 expression strain shows remarkable trypsin inhibitory activity and slightly It showed elastase inhibitory activity.

Each expression system created in the present invention is a prototype, and if further improvement is required in the future, the N-terminal peptide-added Knitz-type domain 1 C-terminal coding region was used to replace synthetic DNA. The Pst I site can be used, and in the case of the Knitz-type domain 2, the same can be done using the ScaI site for the C-terminal code region and the newly provided SphI site for the N-terminal code region. it can.

In addition, the modified DNA was used for pH H305 in which the Kunitz-type domain 1 gene was added as a cassette and pHH306 in which the Kunitz-type domain 2 gene was added as a cassette. It can be used to create a Pichia yeast expression system.

Example 3 UTI (hereinafter referred to as ntntact-rUTI) prepared by genetic recombination in Example 1 and Kunitz type domain 2 (hereinafter, r) of UTI prepared by genetic recombination in Example 2 D2) from Pichia yeast culture supernatant

(1) Purification from culture supernatant

I ntact - r UT I produce yeast culture supernatant, 1 0 k D cut of concentrated approximately 1 0-fold with an ultrafiltration membrane (F iltron OMEGA 10K MINISETTE 75 SQ FT), 50mM Tris, 20mM CaCl 2 containing Dialysis was performed using the solution (PH8.0). The culture supernatant of the rD2 producing yeast was adjusted to pH 8.0 with ΙΝ-NaOH after adding CaCl ₂ to a final concentration of 20 m. Add this preparation to an Anhydrotrypsin-Agarose column (ø1x3.5cm), wash the non-adsorbed fraction with 50raM Τris, 20mM CaC .0.5M NaCl, and elute the adsorbed fraction with 0.2N HC1, 0.2NaCl As a result, Intact-rUTI and rD2 were purified. For the eluted fraction, for Intact-rUTI, fully dialyzed with distilled water and concentrated with Centri-Reb 10.On the other hand, for rD2, adjust the pH to around 8 with 0.1 IN NaOH and use Centr-Rep 3. After concentration, each was stored at -80 ° C. The loading amount on the column was 7,700-12, OOOu as the UTI titer (tribsine inhibitory activity, Reference Example 2), and the activity transfer to the Pass fraction was 1.0-1.7 by Intact-rUTI. % And rD2 were 10.8-16.2%. Final yields were all in the range of 54.6-67.6%. Urine-derived natural UTI was prepared according to the method described in JP-A-5-9200. Also, natural UT

I-derived domain 2 is reported by Hochstrasser et al. CHoppe-Seyler's Z. Physiol, chem.

364.? 1689-1696. (1983)], and the natural UTI was prepared by limited digestion with trypsin.

(2) Analysis of properties

① SDS-PAGE

The purified Intact-rUTI and rD2 were subjected to SDS-PAGE according to the method of Laemmli et al. The three clones of Intact-rUTI show similar migration patterns, and no difference was observed in migration patterns between reducing and non-reducing conditions. won. The same was true for the two clones of rD2. Intact-r UTI showed a main band at 21.3 kD, a broad minor band around 43 kD and a sharp minor band at 19.4 kD. In 02, a single band was shown at 7.31 (0), and the Kunitz type domain 2 derived from native UTI had the same migration position.

② Gel chromatography

Purification Intact—gel filtration chromatography on rUTI and rD2 (column: Tosoh G300 SWXL, eluent: 0.1 M sodium acetate-0.3 M NaC1, pH 6.5, Beckman) Gold system was used). Fig. 23 shows the HPLC-GPC pattern of Intact-rUTI, and Fig. 24 shows the HP LC-GPC pattern of rD2.

③ N-terminal amino acid sequence analysis

N-terminal amino acid sequence analysis was performed on the three bands observed in SDS-PAGE of the Intact-rUTI clone and the 7.3 kD band of rD2. Table 1 shows the results.

table 1

Analysis result of N-terminal amino acid sequence of rUT 〖and rD2 Sample sequence

UTI 43kD A-V-L-P-Q-E-B-E-G-S- 21.3kD A-V-L-P-Q-E-E-E-G-S- 19.4kD E-D-S-C) -Q-L-G-Y-S-

D2 T-V-A-A-C) -N-L-P-I-V-

() 內 does not have a corresponding chromatographic peak for amino acid analysis

Indicates that When the results shown in Table 1 are compared with the primary structure of native UTI, 43 kD and 21.3 kD of Intact-r UTI are complete molecular forms on the amino acid sequence, and 19.4 kD is the N-terminal 22 residues were found to be missing. Further, it was confirmed that rD2 had a sequence of the 78th amino acid and subsequent amino acids according to the cDNA design.

④ Measurement of K i value

The Ki values of purified Intact-rUTI and rD2 with respect to pepsin trypsin and human neutrophil elastase were measured.

The measurement of the Ki value followed the method described below.

After reacting various concentrations of the UTI sample and each enzyme at 25 ° C for 10 minutes, two different concentrations of the substrate were added and the absorbance at 405 nm was continuously measured to reduce the initial reaction rate. I asked. From the obtained results, the Ki value was determined using a Dixon block or an Easson-Stedman block [J.G. Bieth Bull.europ.Physiopath. Resp 1980, 16 (suppl.)]. The reaction buffer was a buffer (pH 8.0) consisting of 0.1 M Tris. LOm CaCh and 0.1% Triton X-100 (hereinafter referred to as buffer A), and the substrate was S-2222 (tryptic). S-2484 (manufactured by kabi) was used for neutrophil elastase. The reaction conditions are as follows.

Ushitoripushin were diluted 20mM C a C 1 _2, with 0.1% tri-ton X- 1 0 0-containing solution (pH 3. 0) 6. 3 X 1 0_ β Μ. To the Ushitoripushin liquid 20 U 1, the UT I sample 8 0 // 1 buffer was varying concentrations diluted 1 0_ ⁸ to 1 0- ⁹ M in Α addition, further buffer A 220 ju 1 or 3 1 0〃 Added one. After reacting at 25 ° C for 10 minutes, 1201 or 30a1 of 5 mM dissolved in distilled water was added to each reaction solution, and the absorbance at 405 nm was measured over time.

Neutrophils Heras evening over Ze _{is, 20mM C a C l 2,} 0. 1 OmM acetate buffer containing 1% Tri ton X- 1 0 0 5 x 1 0 in (pH 4. 5, hereinafter referred to as buffer B) It was diluted with an ⁸ M. The diluted neutrophils Heras evening Ichize solution 20 1, a UT I sample 8 0 pi 1 diluted to various concentrations of 1 0 one ⁵ ~ 1 0- ^beta Micromax with buffer A was added, further buffer A 241 or 2901 was added. 25 of this mixture. Reaction for 10 minutes at C After addition, add 100- 21 or 50〃1 of S-2484 dissolved in 25% DMS- 水 -distilled water at 4 mM to each reaction solution, and measure the absorbance at 405 nm over time. Was measured.

Table 2 shows the results obtained using the Dixon block, and Table 3 shows the results obtained using the Easson-Stedman block.

Table 2 Purification r UTI D2 enzyme inhibition constants

Sample K i (M)

Tribcine

UTI Clone 9-2 1.2 X 10 " ¹¹

Clone 9-7 1.3 X 10 ^'11

Clone 9-8 1.5 X 10 " ¹¹

D2 D2-3 4.5 X 10 " ¹²

D2-5 6.7 X 10 ^'12 natural prison I 1.0Σ 10

Table 3 Purification r Enzyme inhibition constant of UTI D2

Sample K i (M)

Tribcine elastase

UTI clone 9-2 3.1 X 10 ~ ¹⁰ 1.0 X 10

Clone 9-7 3.2 X 10 ~ ¹⁰ 6,8 X 10 Clone 9-8 3.8 X 10 " ¹⁰ 1.0 X 10 r D2 D2-3 4.5∑ 10- ¹¹ 8.5 X 10

D2-5 1.7 I 10 " ¹⁰ 6.2 X 10 Natural UTI 2.1 X 10 -10 3.6 X 10

As shown in Tables 2 and 3, the Ki values of Inta1-rUTrD2 were equivalent to those of native UTI with respect to Tribcine inhibition. However, in the neutrophil elastase inhibition, the Intact-rUTI had a larger Ki value by 2-3 orders of magnitude than the native UTI, and the inhibition was weaker. Moreover, rD2 was found to have the same inhibitory activity as Intact-rUTI against neutrophil elastase, which was generally considered to be involved in Kunitz-type domain 1 inhibition. The natural UTI used as a control was prepared according to the method described in JP-A-5-9200. Example 4 Construction of modified UTI (Ep 1 -UTI) and its Pichia yeast expression system

(1) Modification of Kun i t z-type domain 1 gene

A modified UTI in which a part of the amino acid sequence of Kunitz type domain 1 of UTI was modified was prepared. The modification was performed by replacing the region containing the active site of Kunitz type domain 1 (Met-Gly-Met-Thr-Ser) with (Ile-Ala-Phe-Phe-Pro). The substitution was performed by the site direct mutagenesis method using a U.S.E. mutagenesis kit (manufactured by Pharmacia). Except where noted, the operation was performed according to the protocol attached to the kit.

The Kunitz type domain 1 gene contained on PHH305 created in Example 2 is inserted in the direction opposite to the transcription direction of the lacZ gene of pUC19. Therefore, the mutagenic primer was designed to be on the same chain as the selection primer of the kit. FIG. 25 shows the sequence of the mutage nic primer. The 5 'end of the prepared mutagenic primer was phosphorylated with T4 kinase, and site-directed mutagenesis was performed according to the kit's lip. Note that Sspl / Stul selection primer (manufactured by Pharmacia) was used as the selection primer. The nucleotide sequence of the obtained clone was confirmed, and the plasmid carrying the Kunitz type domain 1 gene into which the desired modification was introduced was designated pT-325.

(2) Categorization of modified UTI gene (Ep1-UTI gene)

The obtained PTK325 was digested with EcoRII and PstI to recover an N-terminal peptide-added modified Kunitz type domain 1 gene fragment lacking the 3 'region. Next, PHH306 was digested with Sphl and EcoRI to recover a Kunitz-type domain 2 gene fragment lacking the 5 'region. Then, a synthetic DNA corresponding to the region between the Sphl sites on Kunitz type domain 2 was prepared from the Pst 1 site on Kunitz type domain 1. These three were ligated to pUC19 digested with EcoRI to obtain PTK332 carrying the modified UT [gene of interest (Epl-UTI gene). (3) Creation of Pichia yeast expression plasmid

The Epl-U expression plasmid pHH334 was constructed by linking the secreted modified UTI gene unit obtained by digesting PTK332 with EcoRI to the EcoR site of the expression plasmid PA0807N (Fig. 26).

(4) Preparation of expression strain and its properties

Epl-UTI expression plasmid PHH334 was digested with a restriction enzyme Stul, and Pichia yeast strain GTS115 was transformed in the same manner as in Examples 1 and 2.

The obtained transformant was inoculated into 5 ml YNB medium and cultured at 30 ° C. for 2 days. This is inoculated into 5 ml of 2 x YP-2% glycerol medium at 10%, cultured at 30 ° C for 2 days, then methanol is added to the medium to a final concentration of 4%, and further cultured at 30 ° C for 5 days did.

The culture supernatant obtained by centrifuging the culture solution was measured for trypsin inhibitory activity and human neutrophil elastase inhibitory activity according to the methods described in Reference Examples 2 and 3. As a result, while exhibiting remarkable trypsin inhibitory activity, the enhancement of human neutrophil elastase inhibitory activity was remarkable as compared with the unmodified [ntact-rUT] shown in Example 2.

(5) Purification of Ep1-1UTI from yeast culture supernatant

After centrifuging the culture supernatant 3 70 Om1 at 700 rpm at 4'C for 30 minutes (TOM YΝο 17 Rotor), centrifuge the obtained supernatant to 0.45 / m membrane ( After passing through MILL I PAK ^R 200 0 MPHL 20 CA 3), the mixture was concentrated to 750 ml using a 10 kD cut ultrafiltration membrane (FI LTRON MINI SETTE ™ OS 0 10 C 0 1). . The UTI titer of the culture supernatant after 0.45 m membrane filtration was 25.6 u / m1 and that of the concentrated culture supernatant was 99.2 u1. To the concentrated culture supernatant after addition of C a C 1 ₂ to a final concentration of 20 mM, adjusted to pH 8. 0 with 1 N-Na_〇_H. The preparation Anhydrotrypsin-Agarose column (02. 0 2. 2 cm) the non-adsorbed fraction was added to 5 0 mM Tr is, 2 OmM C a C 1 2 -containing solution (p H 8. 0) and 5 0 mM After thorough washing with a solution containing Tris, 2 OmM CaCl ₂ and 25 OmM benzamidine (pH 8.0), the adsorbed fraction was eluted with 0.2 N HC 1 and 0.2 M Na C 1. The eluted fraction is collected as 5 OmM Tris, 2 OmM C a C It was dialyzed against a 12-containing solution (pH 8.0) and concentrated with Centri-Reb 10 to obtain a purified product. The UTI titer of the purified product was 2160/1, and the recovery relative to the column loading titer was 50.1%.

The SDS electrophoresis pattern of the purified product showed a main band at 20.9 kD and a minor band at 17.1 kD. 20.9 kD is a complete molecular form in terms of amino acid sequence, and 17.1 kD has 22 amino acids at the N-terminus deleted, and it is considered that sugar chains are added to all molecules. Was.

(6) Characterization of purified Ep 1 -UT I

① Enzyme inhibitor constant (K i) of purified Ep 1 -UT I

The i-values of various purified Ep1-UTI enzymes [trypsin (human, human), plasmin, and neutrophil elastase] were measured according to the following method.

After various and Ep 1 one UT I sample and enzyme concentration was reacted 10 minutes at 25 ^e C, the absorbance of the substrate was added in two concentrations 4 05 nm measured communication铳的, initial reaction rate Was asked. From the obtained results, the Ki value was calculated by the secondary plot of Dixon block or Lineweaver-Burk. The buffer used in the reaction system was Buffer A. The substrate used was S-2222 (manufactured by Kabi) for trypsin, S-2484 (manufactured by Kabi) for neutrophil elastase, and S-2251 (No. 1 for plasmin). (Manufactured by Chemicals).

The reaction conditions are as follows.

Ushitoribushin is 20mM C a C 12, 0. 1 % Tri ton X- 1 0 0, with ρ Η 3. 0 6. 3 x 1 0 - diluted ⁹ Micromax. To the Ushitoribushin liquid 20 1, buffer solution Α in 1 0_ ⁸ ~ 0- ⁸ M a UT I sample 8 0〃 1 made various concentrations diluted in addition, further buffer A plus 220〃 1 or 3 1 0/1 . After reacting at 25 for 10 minutes, 1201 or 301-1 of S-2222 dissolved in distilled water to 5 mM was added to each reaction solution, and the absorbance at 405 nm was measured over time.

Human Toribushin were diluted 20 mM C a C 12, 0. 1% Tri ton X- 1 0 0-containing solution (ρΗ 3. 0) at 1 X 1 0 _ ⁷ Μ. To the Hitotoribushin liquid 20〃 1, a UT I sample 8 0 mu. 1 made various concentrations diluted in buffer Α to 1 0_ ^⁸ ~ 1 0- ⁸ M was added Further, Buffer A was added to 220 u \ or 3101. These were treated in the same manner as in the case of cytrypsin, and the absorbance was measured.

Neutrophil elastase was diluted to 5 × 10 ⁸ M in buffer B. The Hellas diluted evening one peptidase solution 20 1, buffer 1 0_ ⁵ ~ 1 0_ ⁹ M a UT I sample 8 0〃 1 diluted to various concentrations was added in A, 0 24 Buffer A military 1 or 29 0〃1 was added. 25 After reacting at 50 ° C for 10 minutes, add 1— or 4 5 of S‐2484 dissolved in 25% DMSOZ distilled water to 4 mM to each reaction solution, and add the absorbance at 405 nm over time. It was measured specifically.

Plasmin was diluted to ⁸ × 10 ⁸ M in buffer B. The plasmid down liquid 4 0/1 diluted buffer A at 1 0- ⁵ ~ 1 0_ ⁸ M a UT I sample 8 0 1, diluted to various concentrations was added, further the buffer A 1 8 0/1 added Was. After reacting at 25 ° C for 10 minutes, S-2251 dissolved in distilled water at 5 mM or 2.5 mM was added to each reaction solution, and the absorbance at 405 nm was measured over time. .

Table 4 shows the Ki values of purified Ep1-1UTI together with the values of Intact-rUTI and native UTI similarly measured. In Table 4, the values marked with * were obtained by the secondary plot of Lineweaver-Burk, and the other values were obtained by the Dixon plot.

Enzyme inhibitor constant (K i :: nM) Enzyme

Natural UTI Intact-rliTI Epl-UTI Tribcine (maggot) 0.21 0.31 0.38 Tribcine (human) 3.9 * 6.3 * 6.4 * Plasmin (human) 770 460 460 600 Neutrophil elastase 3.6 1000 0.57 Table 4 As shown in Fig. 5, for tribine and plasmin, the inhibitory activity of which is attributed to the Kunitz-type domain 2 region of UTI, the Ki value is the same as for native UTI,

Intact—rUT I and Ep 1—UT I had almost the same values, whereas neutrophil elastase, whose inhibitory activity is attributed to K unitz-type domain 1, had Knitz-type domain 1 The Ep 1 -UT I modified at the active center had an order of magnitude lower than that of the native UTI and a 4-order-one Ki value lower than that of Intact-rUT I, indicating an increase in inhibitory capacity.

②Evaluation of oxidation sensitivity

Ep 1-UTI solution (2 X 10 " ⁸ M) diluted with buffer A and natural UTI solution (1 X 1 (T ⁶ NO each 801) 1. Chloramine T (manufactured by Nacalai Tesque) solution, which was serially diluted 3-fold up to 67 M, was added at 120/1, and allowed to react at room temperature for 10 minutes.Then, 10 OmM dissolved in water was added to each reaction solution. The reaction was stopped by adding L-methionine (Nacalai Tesque, Inc.) 衝液B neutrophils Elastica dissolved in evening ^{Ichize. (5 X 1 0 - 8} M) 20〃 1 was added to 25 "C-Te reacted for 10 minutes then to each reaction, 25% DMS And 1 mM of 4 mM S-2484 (manufactured by Kabi) dissolved in ◦ was added, and the initial reaction rate was measured with a spectrophotometer (405 nm).

FIG. 27 shows the effects of Ep1-UTI oxidized by chloramine T and native UTI on human neutrophil elastase inhibition. The residual inhibition rate in Fig. 27 is the inhibition rate of chloramine T non-oxidized inhibitor when the inhibitory rate is defined as the ratio of the decrease in the initial reaction rate when the inhibitor is added to the initial reaction rate when the inhibitor is not added (). The ratio (%) of the inhibition rate of the chloramine T oxidation inhibitor with respect to is shown. As shown in FIG. 27, when the concentration of the reacted chloramine T was increased, neutrophil elastase inhibitory activity was reduced in the urinary native UTI due to chloramine T oxidation, whereas Ep1-1UT In I, the inhibitory activity did not decrease. From the above, by substituting a part of the amino acid sequence of UTI with another amino acid, it is possible to maintain the same trypsin inhibitory activity and brassin inhibitory activity as natural UTI and Intact-r UT1. On the other hand, the neutrophil elastase inhibitory activity could be increased by one order as compared to natural UTI and four orders as compared to Intact-rUTI, and the effect of the modification was observed.

Ep 1—UTI is a methionine at position 36 from the N-terminus of urine-derived natural UTI (see Figure 5) and is position 38 from the N-terminus of urine-derived natural UTI (see Figure 5). The methionine is replaced by phenylalanine, and this substitution is expected to improve oxidation resistance.

Chloramine T is known to oxidize methionine to methionine sulfoxide in neutral or weakly acidic conditions, but as shown in Figure 27, the neutrophil elastase inhibitory activity of Ep1-UTI It was confirmed that compared to UTI, it was not affected by chloramine T oxidation. It is known that neutrophils release active oxygen together with elastase in the field of inflammation, but EP 1 -UTI is expected to work stably even in such a field. You. Example 5 Preparation of N-terminal 21 amino acid deletion modified UTI (hereinafter referred to as Ep 1 -d 21) and its Pichia yeast expression system

(1) Categorization of Ep1-d21 gene

PTK332 obtained in Example 4 was digested with Ec052I and EcoRI, and added to the N-terminal side of the EcoRI fragment of the vector DNA and the Knitz-type domain 1. An Eco 52 I-EcoRI fragment of the UTI gene lacking the 5 'terminal region encoding the amino acid region was recovered.

For the SUC2 signal, the synthetic DNA shown in FIG. 15 was used. In addition, the region from the 5 'end, which encodes the 21 amino acid region added to the N-terminal side of the Knitz-type domain 1 to the Ec052 I site, originally contains the corresponding synthetic DNA. It was prepared by eliminating the vull site (Figure 28). By linking these four members, a plasmid pHH336 carrying the Ep1-d21 gene was prepared (FIG. 29).

(2) Preparation of Pichia yeast expression plasmid

The Ep1-d21 gene obtained by digesting pHH336 obtained in (1) with EcoRI was ligated to the EcoRI site of PAO 807 N, and the expression plasmid PHH339 was ligated. Created (Figure 30).

(3) Preparation of expression strain and its properties

Ep1-d21 expression plasmid pHH339 was digested with restriction enzyme StuI, and Pichia yeast strain GTS115 was transformed in the same manner as in Examples 1 and 2.

The obtained transformant was inoculated into 5mlYNB medium and cultured for 2 days at 30 ^e C. This 2 days after culture in 10% inoculated 30 ^e C to 2xYP-2% glycerol medium of 5 m 1, methanol was added to the medium to a final concentration of 4%, and further cultured for 5 days at 30 ^e C .

The obtained culture supernatant showed remarkable human neutrophil elastase inhibitory activity as compared with the unmodified Intact-rUTI obtained in Example 1.

(4) Purification of Ep1-d21 from yeast culture supernatant

After 9000 r pm, 30 minutes of culture supernatant 2000 m 1 at 4'C (TOM Y No 9 Ro tor ) centrifugation, the resulting supernatant 0. 45 m membrane (MI LL I PAK ^R 20 0 Milipore MPHL 2 0 CA 3) After concentrating the filtrate to 1 0 0 m 1 in 1 0 kD cutlet Bok ultrafiltration Filtration membrane (FI LTRON MINI SETTE ™ OS 0 1 0 C 0 1), from 5 0 mM T ris, 2 0mM C a C 1 2 The resulting solution was dialyzed against 10 L of a solution (pH 8.0). The UTI titer of the culture supernatant after concentration was 23 uZm 1. The concentrated culture supernatant after dialysis, Anhydrotrypsin-Agarose column (02. 0 X 2. 2 cm) the non-adsorbed fraction was added to 5 OmM Tr is, 2 0 mM C a C 1 2 -containing solution (pH 8. 0) and 5 0mM Tr is, 2 Om C a C 1 2, 2 5 after sufficient washing OmM benzamidine-containing solution (pH 8. 0), 0. 2N HC 1 adsorbed fraction, 0. 2M Na C 1 containing Eluted with solution. The eluted fraction was dialyzed against distilled water and concentrated with Centriprep 10 to obtain a purified product. The UTI titer of the purified product was 244 4 / m1.

The SDS electrophoresis pattern of the purified product showed a main band at 17.7 kD. It was considered that a sugar chain was added.

(5) Characterization of purified Ep1-d21

(1) Enzyme inhibitor constant (K i) of purified Ep 1-d 21

The Ki values of various purified Ep 1 -d 21 enzymes [trypsin (pishi, human), human plasmin, and human neutrophil elastase] were determined.

Specifically, the Ki value was determined according to the following method.

After reacting Ep 1 -d 21 samples at various concentrations with the enzyme at 25'C for 10 minutes each, the substrate was added at two concentrations and the absorbance at 405 nm was continuously measured. The speed was determined. From the obtained measurement results, Ki was obtained by Easson-Stedman block (JG Bieth Bull.europ.Physiopath.resp 1980.16 (suppl.) 183-195), Dixon plot or Lineweaber-Burk secondary plot. The value was determined. Buffer A was used as a buffer for the reaction system. The substrate used was S-2222 (Kabi) for trypsin, S-2484 (Kabi) for neutrophil elastase, and S-2251 for brasmin. (Manufactured by Daiichi Kagaku). The reaction conditions are as follows.

ゥ Citribulin contains 2 OmM Ca C 12, 0.1% Triton X—100 Diluted ⁹ M - 6. 3 x 1 0 with a solution (pH 3. 0). To the Ushitoripushin liquid 2 0 1 Buffer E p 1 and various concentrations diluted 1 0 one ⁸ ~ 1 0- ^beta Micromax in A - d 2 1 sample 8 0 1 was added, further buffer A 2 2 0 1 Or 3 1 0〃 1 added. After reacting at 25 for 10 minutes, add S-222, dissolved in distilled water to 5 mM, to each reaction solution, add 121 or 30/1, and measure the absorbance at 405 nm over time. It was measured.

Human Toribushin is 2 0 mM C a C 1 2 , 1 X 1 0 with 0.1% tri-ton X- 1 0 0-containing solution (pH 3. 0) - were diluted into ⁷ M. To the Hitotoribushin liquid 2 0 1 Buffer A at ^{^{1 0- 8 ~ 1 0 - 9}} E p 1 and various concentrations diluted in M - d 2 1 sample 8 0 1 was added, further buffer A 2 2 0 / 1 or 3 1 0〃 1 was added. These were treated in the same manner as in the above-mentioned caseitribins, and the absorbance was measured.

Neutrophils Heras evening Ichize was diluted to 5 XI CT ^e M with buffer B. A medium favorable diluted elastase solution 2 0 [/ 1, buffer 1 0 ^5-1 0 A - diluted to various concentrations in ⁹ M E pl - d 2 1 sample 8 0 1 was added, Buffer A Was added to 240 41 or 2901. After reacting at 25 for 10 minutes, add 100 / zl or 50/1 of S-2484 dissolved in 4 mM 25% DMS-Z distilled water to each reaction solution, and add 4 The absorbance at 05 nm was measured over time.

Plasmin was diluted to ⁸ × 10-8 M in buffer B. The Burasumin liquid 4 0〃 1 diluted buffer A at ^{^{1 (T 5 ~l 0_ 8 E}} p 1 was diluted to various concentrations Micromax - d 2 1 test body 8 0 1 was added, further buffer A 1 8 After adding 10 minutes at 25, the reaction mixture was added with S-2251 dissolved in distilled water at 5 mM or 2.5 mM, and added to each reaction solution. The absorbance at nm was measured over time.

Table 5 shows the Ki values of purified Ep 1 -d 21 for various enzymes [trypsin (human, human), blasmin, and human neutrophil elastase] measured under the above measurement conditions. The values are shown together with the Ki values of Intact-rUTI, natural UTI and Ep1-UTI. In Table 5, the values marked * 1 were determined by Easson-Stedman plots, the values marked * 2 were determined by the Lineweaver-Burk secondary plot, and the other values were determined by Dixon plots. Enzyme inhibitor constant (Κ i: ηΜ)

aft

Drunk cord

Natural UTI Intact-rUTI Epl-UTI Epl-d21 Tribcine (ゥ) 0.010 Trypsin (human) 3.9 · ² 6.3 * ² 6.4 * ² 0.79 * ² ο

ο

Brassin (Human) 770 460 <· — 600 990 Neutrophil elastase 3.6 1000 0.145 0.093

o

As shown in Table 5, with respect to tribcine and plasmin inhibition, the Ki value of UTI (Intact-rUTEpl—iUTI, Ep1-d21) created by genetic recombination was the same as that of native UT. I ranged from 1.6 to 1.6 times that of I, but with regard to neutrophil elastase inhibition, Ep 1— 1/25 of UTI in native UTI and 1 in Ep1-d21 in Ep1-d21. The Ki value was remarkably reduced to 39, and a clear increase in inhibitory capacity was observed.

②Evaluation of oxidation sensitivity

Ep 1-d21 and natural UTI each diluted to 5 M with 5 OmM phosphoric acid buffer solution (pH 7.0) and natural UTI each 25], and the same buffer continuously from 2 OmM to 1.28 M 5 Chloramin T (manufactured by Nacalai Tesque, Inc.) was added in a concentration of 25 × 1 and reacted at room temperature for 20 minutes. Then, after dilution 9. 4 X 1 0- ⁸ M in each reaction to 20 Omm of L one Mechionin aqueous 50 i 1 added to stop the reaction buffer A, to the 40 1, buffer A110 u \, and buffer B at 2. human neutrophils diluted 5 X 1 0- ⁷ M The sphere elastase was added in 201 and reacted at 25 for 10 minutes. Next, 50〃1 of 2 mM S-2484 (manufactured by Kabi) dissolved in 25% DMS0 was added to each reaction solution, and the mixture was reacted at 25 for 5 minutes, followed by adding 701 of a 20% acetic acid solution and reacting. Was stopped and the absorbance at 405 m was measured.

Table 6 shows the treatment concentrations of chloramine T in which human neutrophil elastase activity remains at 80% or more in Ep1-d21 and native UTI. Natural UTIs are lost at relatively low concentrations. On the other hand, Ep 1—d21 uses low concentrations of chloramin T, which inactivates natural UTIs as well as Ep1-1 UTIs. Oxidation did not deactivate at all, and an apparent increase in oxidation resistance was observed.

Table 6 Chloramine treatment concentration Native UTI <3.2 uU

E ρ 1 -d 21 <400 m Example 6 Comparison of expression levels of E p 1—UTI expressing strain and Ep 1 -d 21 expressing strain in test tube culture

The culture supernatant of the Ep1-UTI expression strain obtained in Example 4 (4) and the culture supernatant of the Ep1-d21 expression strain obtained in Example 5 (3) were The expression level of UTI was measured by a sandwich ELISA using an anti-rUTI antibody (Reference Example 1). Savior peroxidase mono-avidin D (VECTOR LA BORATOR IES) was used as the chromogenic enzyme, and peroxydase color-developing kit 0 (Sumitomo Bei-Client) was used as the chromogenic substrate according to the supplier's recommendations. used. The general procedure followed a book. The measurement was performed using Titertec Multiscan MCCZ340 MKII (manufactured by Flow Laboratories), and ΔS0FT (vers.2.12F, manufactured by BioMetallies) was used for data analysis. As a result, each modified UTI of 0. TmgZL was detected in the culture supernatant of the Ep1-UTI expression strain, and 0.1 TmgZL in the culture supernatant of the Ep1-d21 expression strain. On the other hand, a difference of more than 14-fold was observed in the expression level depending on the presence or absence of the N-terminal peptide of UTI comprising 21 amino acid. In addition, as shown in Table 4, the enzyme-inhibiting activities of both modified UTIs are equivalent. It was suggested to contribute to the increase.

Example 7 Comparison of expression levels by high-density flask culture

In Example 6, Ep1-UTI having the N-terminal peptide of UTI consisting of 21 amino acids was expressed in a larger amount by genetic engineering compared to Ep1-d21 not having it. It was suggested that To confirm this, flask cultivation (high-density flask cultivation) was performed with increased bacterial cell density during methanol induction, and the expression levels were compared.

Example 4 (4) obtained in Ep 1-UT I expressing strain and Examples 5 (3) E obtained in p 1 - a d 21 expression strain was inoculated into ΥΝΒ medium 2m 1 30 ^e C For two days. This was inoculated into 100 ml of 3 XYP—2% glycerol medium (3% yeast extract, 6% eptone, 2% glycerol) in 1 ml portions, and cultured at 30 ° C for 2 days. The turbidity at O nm (OD <o.) Was measured.

After harvesting from the culture, each OD _eo . The suspension was suspended in 2XYP-4% methanol medium (2% Yeast Extract. 4¾ Peptone, 4¾ methanol) so that the value was about 250. Place 40ml of these in a 300ml Erlenmeyer flask with baffle. The cells were cultured at 150 rpm for 72 hours at C. The turbidity was measured every 24 hours, and the culture supernatant for measuring the expression level was collected and stored frozen in TC (TC).

After completion of the culture, the culture supernatant stored frozen was thawed spontaneously at room temperature, and the expression level of each modified UTI was measured by the sandwich ELISA method as in Example 6. Table 7 shows the turbidity and expression level for each hour. W

Table 7

High-density flask culture of Eg! -UT I and E_p1 -d21

Ohr 24hr 48h'r 72hr

ODeoo ODgoo expression level ODeoo expression level ODfioo expression level Modified UTI (mg / L) (mg / L) (mg / L)

Epl-UTI 295 365 193 329 197 305 236

Epl-d21 273 353 2.9 337 3.6 318 3.7

236 mg ZL was detected in the culture supernatant of the Ep1-UTI expressing strain, and 3.7 mgZL of each modified UTI was detected in the supernatant of the Ep1-d21 expressing strain. That is, there is a 63-fold difference in the expression level depending on the presence or absence of the N-terminal peptide of UTI consisting of 21 amino acids, indicating that the N-terminal peptide of UTI contributes to the increase in the expression level in gene engineering techniques. Mosquito, the results were even more remarkable than the test tube culture of Example 6.

One of the advantages of using Pichia yeast as a host in an expression system is that the density of cells can be increased under optimally controlled culture conditions. Under the high-density conditions of the cells shown in this example, the E1-UTI expression strain having a 21 amino acid UTI N-terminal peptide was transformed into an Ep1-d21 expression strain without it. The remarkably superior expression level suggests that the expression level can be further increased by examining the culture conditions in a controllable culture tank.

Reference Example 1 Preparation of affinity-purified polyclonal anti-rUTI antibody

The heron anti-blood immunized with natural UTI was subjected to ammonium sulfate decay, and Ig fractions were collected. This is adsorbed to an affinity column in which natural UTI is immobilized on FMP-activated Cell mouth Fine (manufactured by Seikagaku Kogyo Co., Ltd.). After washing, the column was eluted with 0.1 M glycine hydrochloride (pH 2.5). In addition, if necessary, biotinylation was performed according to a conventional method.

Reference Example 2 Method for measuring UTI titer (trypsin inhibitory activity) 200 u \ of UTI sample diluted to about 50 u / ml, reaction buffer (0.1 Tris-HCl, 20 mM CaC, 1.5 mg / ml Gelatin, pH 8.0) 1.6 mK diluted to 100 g / ml. After mixing with trypsin 200 \ and leaving it to stand for 5 minutes, lmg of 5 mg / ml L-BApNa (N-hy-bengoytri L-Arginine-p-Nitroanilide) was mixed and allowed to react for exactly 5 minutes. The reaction was stopped by mixing 1 ml of 20% acetic acid with the reaction solution, and the absorbance at 405 nm was measured. UTI was diluted with the above reaction buffer, and tribcine was diluted with 20 mM CaCl ₂ , 1.5 mg / ml Gelatin, pH 3.0.

Reference Example 3 Measurement of human neutrophil elastase inhibitory activity

Add 1 \ 0 fi \ of buffer A to 40 pi 1 of the inhibitor solution diluted to 9.4 X10_ ⁸ M with buffer A, and add 2.5 μl (20 μl of neutrophil elastase diluted to Γ ^{7.) With} buffer B. 1 added was allowed to stand 25 ° C 30 minutes. then 2 (manufactured by Kabi Co.) Om S- 2484 / / 25% DMSO for 5 0 p. 1 added 25 ^e C after 5 minutes reaction, 20% acetic acid solution The reaction was stopped by adding 70 1 and the absorbance at 405 nm was measured.

Claims

The scope of the claims

1. An expression vector for a yeast belonging to the genus Pichia containing a DNA having a nucleotide sequence encoding the amino acid sequence of the kunitz type domain 1 of urinary trypsin inhibitor.

2. An expression vector for Pichia yeast containing a DNA having a nucleotide sequence coding for the amino acid sequence of Knitz type domain 2 of urinary trypsin inhibitor.

3. An expression vector for a yeast belonging to the genus Pichia, which comprises a DNA having a nucleotide sequence encoding an amino acid sequence of a urinary trypsin inhibitor.

4. A yeast of the genus Pichia transformed by the expression vector for yeast of the genus Pichia according to any one of claims 1 to 3.

5. culturing the yeast belonging to the genus Pichia according to claim 4 to produce a protein having an amino acid sequence of at least one Kunitz type domain of a urinary trypsin inhibitor, and collecting the protein from the resulting culture A method for producing a protein having at least one kind of Kunitz-type domain amino acid sequence of a urinary trypsin inhibitor.

6. A novel polypeptide having at least the amino acid sequence of the following formula I:

Cys-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-Cys-

Ile-Al a-Phe-Phe-Pro-Arg-Tyr-Phe-Tyr-Asn-

Gly-Thr-Ser-et-Ala-Cys-Glu-Thr-Phe-Gln-

Ty r-G 1 y-G 1 y-Cys-Me t-G 1 y-Asn-G 1 y-Asn-Asn- (Formula I)

P e-Val-Thr-Glu-Lys-Glu-Cys-Leu-Gln-Thr-

Cys

7. The novel polypeptide according to claim 6, wherein the polypeptide has the following amino acid sequence [II].

Thr-Val-Ala-Ala-Cys-Asn-Leu-Pro-Ile-Val

Arg-Gly-Pro-Cys-Arg-Ala-Phe-I le-Gln-Leu-

Trp-Ala-Phe-Asp-Ala-Val-Lys-Gly-Lys-Cys-

Val-Leu-Phe-Pro-Tyr-Giy-Gly-Cys-Gln-Gly-

Asn-Gly-Asn-Lys-Phe-Tyr-Ser-Glu-Lys-Glu- (Formula 111)

Cys-Arg-Glu-Tyr-Cys-Gly-Val-Pro-Gly-Asp-

Gly-As -G 1 u-G 1 i shi eirLeu-Arg-Phe

8. A novel polypeptide characterized by having the amino acid sequence of the following formula IX.

Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr-Ser-

Ala-Gly-Pro-Cys- 11 e-Ala-Phe-Phe-Pro-Arg-

Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-et-Ala-Cys-

Glu-Thr-Phe-Gln-Tyr-Gly-Gly-Cys-et-Gly-

Asn-Gly-Asn-Asn-P e-Val-Thr-Glu-Lys-Glu-

Cys-Leu-Gln-Thr-Cys-Arg-Thr-Val-Ala-Ala-

Cys-Asn-Leu-Pro-I le-Val-Arg-Gly-Pro-Cys-

Arg-Ala-Phe-I le-Gln-Leu-Trp-AIa-Phe-Asp-

Ala-Val-Lys-Gly-Lys-Cys-Val-Leu-Phe-Pro-

Tyr-Gly-Gly-Cys-Gln-Gly-Asn-Gly-Asn-Lys-

Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu-Tyr- (Formula IX)

Cys-Gly-Val-Pro-Gly-Asp-Gly-Asp-Glu-Glu- and eu- and eu-Arg_Phe

9. Claim 6 characterized in that it has an amino acid sequence of the following formula II on the N-terminal side. 9. The novel polypeptide according to any one of claims to 8.

Ala-Val-Leu-Pro-Gln-Glu-Glu-Clu-Gly-Ser

Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr- (Formula II) Lys

10. A novel polypeptide having an amino acid sequence represented by the following formula: [V]

Ala-Val-Leu-Pro-Gln-Giu-Glu-Glu-Gly-Ser-

Gly-Gly-Gly-Gln-Leu-Val -T r-G lu-Val-Thr-

Lys-Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr-

Ser-Ala-Gly-Pro-Cys-I le-Ala-Phe-Phe-Pro-

Arg-Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-et-Ala-

Cys-Glu-Thr-P e-Gln-Tyr-Gly-Gly-Cys-Met-

Gly-Asn-Gly-Asn-Asn-Phe-Val-Tr-Giu-Lys-

Glu-Cys-Leu-Gln-Thr-Cys-Arg-Thr-Val-Ala-

Ala-Cys-Asn-Leu-Pro-l le-Val-Arg-Gly-Pro-

Cys-Arg-Ala-Phe-I le-Gln-Leu-Trp-Ala-Phe-

Asp-Ala-Val-Lys-Gly-Lys-Cys-Val-Leu-Phe-

Pro-Tyr-G 1 y-G 1 y-Cys-G 1 n-Gly-Asn-Gly-Asn-

Lys-Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu- (Formula IV)

Tyr-Cys-Gly-Val-Pro-Gly-Asp-Gly-Asp-Glu-

Glu—Sheu—Sheu—Arg—Phe

11. The novel polypeptide according to any one of claims 6 to 10, which has an inhibitory activity on neutrophil elastase.

1 2. Natural type urinary trypsin inhibitor with inhibitory activity against tribcine The novel polypeptide according to any one of claims 6 to 10, wherein the inhibitory activity on neutrophil elastase is enhanced as compared with the evening.

1 3. A DNA having a base sequence encoding the novel polypeptide according to any one of claims 6 to 12.

1 4. A vector comprising the DNA according to claim 13.

1 5. A transformant transformed with the vector according to claim 14.

1 6. Culturing the transformant according to claim 15 to produce the novel polypeptide described in any one of claims 6 to 12, and obtaining the novel polypeptide from the obtained culture. The method for producing a novel polypeptide according to any one of claims 6 to 12, wherein the peptide is collected.

1 7. Transformation with a vector in which a nucleotide sequence encoding the amino acid sequence of the following formula (II) is ligated to the 5 'end of the nucleotide sequence encoding the amino acid sequence containing the active site of the protease inhibitor. A method for enhancing expression of a protease inhibitor, which comprises culturing the transformed transformant.

Ala-Val-Leu-Pro-Gln-Glu-Glu-Glu-Gly-Ser- (Formula)

Gly-Gl yG ly-Gl n-Leu-Va 1 -Thr-G 1 u-Va 1 -Thr- ys

Amendment * Claims

"I 9 9 fi rod January 16 R (16.0.1.96) H International Affairs Bureau S: Application: The first request Kilffi 17 was filed; new, claim 1 8 has been added; other claims remain unchanged (page 1)]

The novel polypeptide according to any one of claims 6 to 10, wherein the inhibitory activity on neutrophil elastase is enhanced as compared to that of the polypeptide.

13. A DNA comprising a nucleotide sequence encoding the polypeptide according to any one of claims 6 to 12.

14. A vector comprising the DNA according to claim 13.

15. A transformant transformed with the vector according to claim 14.

16. The transformant according to claim 15 is cultured to produce the novel boriveptide according to any one of claims 6 to 12, and the novel polypeptide is obtained from the obtained culture. The method for producing a novel polypeptide according to any one of claims 6 to 12, wherein the method is performed.

17. After correction, a base sequence encoding the amino acid sequence of the following formula (II) is linked to the 5 'end of the nucleotide sequence encoding the amino acid sequence containing the active site of the protease inhibitor. And culturing the transformed yeast in the yeast expression vector.

A 1 a-Ya I -Leu-Pro-Gln-G 1 u-Gl u-Gl u-Gly-Ser-, 11)

Gly-Gly-G 1 y-CIn-Leu-Va 1 -Thr-Gi u-Val-Thr-

18. (Addition) The method for enhancing expression of protease inhibitor Yuichi according to claim 17, wherein the yeast expression vector is an expression vector for Pichia yeast and the yeast is Pichia yeast.

63 Papers captured (Article 19 of the Convention)