JPH0680697A - Natural type polypeptide having activity to inhibit human neutrophile esterase and medical preparation containing the same - Google Patents

Abstract

PURPOSE:To provide an esterase-inhibiting polypeptide or its homopolymer exhibiting an esterase-inhibiting activity and a cathepsin G-inhibiting activity and substantially not exhibiting a trypsin-inhibiting activity. CONSTITUTION:The esterase-inhibiting polypeptide having an amino acid sequence represented by 50 amino acid residues corresponding to the Arg<58>-Ala<107> of the amino acid sequence of SLPI, an amino acid sequence represented by 49 amino acid residues corresponding to the Arg<59>-Ala<107>, their part having the esterase-inhibiting activity, or the biologically equivalent amino acid sequence, or their homopolymers, and their production method, medical preparations containing the substances, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a natural polypeptide having human neutrophil elastase inhibitory activity, a pharmaceutical preparation containing the same, and the like. More specifically, an elastase-inhibiting activity polypeptide having an amino acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2 or an amino acid sequence biologically equivalent to the same, a homopolymer thereof, a method for producing the same, and a therapeutic effect thereof It relates to a pharmaceutical formulation consisting of an amount and a pharmaceutically acceptable carrier.

[0002]

2. Description of the Related Art Chronic bronchitis, diffuse panbronchiolitis, bronchiectasis, emphysema, ARDS, bacterial pneumonia, bronchial asthma and the like are diseases in which neutrophils are increased in sputum and in bronchial and alveolar lavage fluid. Known (Am. Rev. Respir.
Dis., 132 , 417, 1985; Am. Rev. Respir. D
is., 142 , 57, 1990), and thus elastase is released from neutrophils that migrate into the lungs and bronchi (Am. Rev. Respir. Dis., 141 , 689, 19).
90), therefore, it has been reported that high concentration elastase derived from neutrophils is detected in the respiratory tract fluid or purulent sputum of such inflammatory respiratory diseases (Am. Rev.
Respir. Dis., 120 , 1081, 1979; The New
England Journal of Medicine, Jan. 22, 192, 1
981).

Neutrophil proteases are originally foreign foreign substances,
Although it has a role as a biological defense mechanism against bacteria, viruses, etc., when neutrophil protease is present in excess, it attacks itself and the lung matrix is degraded (J. Biol. Chem., 255 , 8848, 1980; J.
Biol. Chem., 255 , 12006, 1980).

[0004] It is the protease inhibitor group that the living body prepares for this excess protease. However, when the neutrophil protease and protease inhibitor are out of balance and the neutrophil protease becomes excessive, the lung matrix becomes It is thought that the disease will become serious if injured and such a condition continues (Am. Rev. Res
pir. Dis., 132 , 417, 1985; Am. Rev. Respi
r. Dis., 129 , 735, 1984; Am. Rev. Respir.
Dis., 142 , 57, 1990; Annals of Internal M
edicine, 111 , 957, 1989).

Human leukocyte elastase inhibitory protein (Secret
ory Leukoprotease Inhibitor (hereinafter referred to as SLPI) is a protease inhibitor that protects the body from excessive neutrophil elastase mainly in the upper respiratory tract by being produced by airway secretory gland cells, but is excessively present in the local inflammation. The mode of degradation of SLPI by neutrophil elastase has not been elucidated.

SLPI is also present in human saliva, nasal discharge, semen, uterine mucus, etc., and is isolated from parotid gland secretion.
The amino acid sequence of PI was analyzed (Proc. Natl. Aca
d. Sci. USA, 83 , 6692, 1986;
No. 501291), and the structure of this protein gene has been isolated from the human parotid gland gene library (Nucl. Acid. Res. 14 , 7883, 198).
6; Special Table Sho 62-501262). However, SLPs found in human saliva, nasal discharge, semen, uterine mucus, etc.
The mode of degradation of I has not been elucidated.

SLPI has not only an inhibitory activity against chymotrypsin-like enzymes such as human neutrophil elastase, human cathepsin G, human pancreatic elastase and human chymotrypsin, but also a physiologically important trypsin-like enzyme such as trypsin. have. Due to this trypsin-like enzyme inhibitory activity, SLPI has been difficult to be used as a therapeutic drug for human diseases, despite having excellent elastase inhibitory activity and cathepsin G inhibitory activity. For this reason, the SLPI carboxy-terminal polypeptide obtained by removing the first half of SLPI and preparing only the second half by genetic engineering is S
It has already been confirmed that LPI has substantially no trypsin inhibitory activity while maintaining the elastase inhibitory activity (Biotech, 7 , 55, 1989; Protein Engine).
ering, 3 , 215, 1990; WO89 / 06239).

However, although these SLPI carboxy-terminal polypeptides have been successful in eliminating the trypsin-like enzyme inhibitory activity that is expected to lead to side effects, when they are applied to therapeutic preparations. , May cause new problems. Because all of the SLPI carboxy-terminal polypeptides reported so far are genetically engineered, so to speak, artificial non-naturally occurring polypeptides, when used in humans as a preparation, antigenicity occurs and antibody production occurs. Is a concern.

When an antibody against a polypeptide preparation is produced, the effect of the preparation is inhibited by the antibody and not only the drug effect disappears but also there is a risk that a rapid antigen-antibody reaction occurs in a living body. Administration of the same formulation to patients will not be possible after antibody production.

[0010] On the other hand, it is known that humans are particularly likely to produce antibodies against non-natural polypeptides, and this risk is high. The previously reported non-natural SLPI carboxy-terminal polypeptides are used as preparations. Of course, you should avoid doing it.

On the other hand, the naturally-occurring polypeptide which already exists in the living body is, so to speak, a self-polypeptide,
The production of antibodies is not a concern, and it is clearly an excellent formulation for human administration.

[0012]

Therefore, it has excellent elastase inhibitory activity and cathepsin G inhibitory activity, does not exhibit trypsin inhibitory activity substantially, and when used as a preparation, there is a risk of developing antigenicity. There is a need to provide a non-elastase inhibitory protein.

The mode of degradation of SLPI by high-concentration neutrophil elastase locally present in inflammatory respiratory disease has not been elucidated, but the fragment degraded by neutrophil elastase is a natural polypeptide present in vivo. That is, it is a self-polypeptide and the production of antibodies is not a concern.

Although the mode of degradation of SLPI existing in saliva has not been elucidated, the fragment decomposed by the enzyme in saliva is a natural polypeptide existing in vivo, that is, a self-polypeptide, and the production of antibodies is No worries.

Accordingly, the present inventors have made diligent efforts to provide such an elastase inhibitory protein, and as a result, natural SLPI produced by a genetic engineering method undergoes specific limited degradation by the enzyme of neutrophil elastase. thing,
The polypeptide that has undergone this limited degradation, that is, the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence biologically equivalent thereto has excellent elastase inhibitory activity, cathepsin G inhibitory activity, and trypsin inhibitor The inventors of the present invention have reached the present invention by finding that they exhibit substantially no activity.

Further, natural SLPI produced by a genetic engineering method undergoes specific limited degradation by an enzyme in normal human saliva, and the polypeptide subjected to this limited degradation, that is, the amino acid sequence represented by SEQ ID NO: 2. Further, the present invention was found by finding that a polypeptide having an amino acid sequence biologically equivalent thereto has excellent elastase inhibitory activity, cathepsin G inhibitory activity, and substantially does not show trypsin inhibitory activity. Is.

Conventionally, straight chain SL carboxymethylated
It has been reported that the PI was enzymatically limitedly decomposed (Proc. Natl. Acad. Sci. USA, 83 , 6692).
-6696, 1986). However, the SLPI used in this case is a non-natural SLPI having no inhibitory activity.
Of course, the resulting fragment is also a non-natural SLPI, and its elastase inhibitory activity, cathepsin G inhibitory activity, trypsin inhibitory activity and antigenicity are not described or suggested.

[0018]

That is, the present invention provides an elastase-inhibiting activity polypeptide having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof having an elastase inhibitory activity, or an amino acid sequence biologically equivalent thereto. Peptide or its homopolymer, the following formula [I] AX-B ... [I] (wherein A represents a carrier protein; B represents the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or elastase inhibitory activity) Represents a protein of interest comprising an elastase-inhibiting activity polypeptide or a homopolymer thereof having a part thereof or a biologically equivalent amino acid sequence thereof; and X is a chemical method or organism under the condition that the protein of interest is not denatured. Represents a cross-linked polypeptide having an amino acid sequence cleavable by biological methods.) Fused protein, method for producing such elastase inhibitory activity polypeptide or homopolymer thereof, gene encoding carrier protein, amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof having elastase inhibitory activity A gene encoding an elastase-inhibiting activity polypeptide having an amino acid sequence biologically equivalent to, a cross-linking peptide having an amino acid sequence cleavable by a chemical method or a biological method under conditions that do not denature the target protein, or a A fusion protein gene linked via a gene encoding a homopolymer, a fusion protein gene-expressing plasmid or recombinant microbial cell containing the fusion protein gene, and a therapeutically effective amount of the elastase inhibitory activity polypeptide or a homopolymer thereof. Medically allowed Pharmaceutical formulation consisting of carriers, diseases caused by neutrophil over-activation, among others, for example, such as elastase and / or cathepsin G, is a therapeutic agent for a disease caused by neutrophils release proteases.

Therefore, the present invention provides an elastase-inhibiting polypeptide or a homopolymer thereof having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof having an elastase-inhibiting activity or an amino acid sequence biologically equivalent thereto. I will provide a.

In the present invention, the "biologically equivalent amino acid sequence" means the elastase inhibitory activity and the cathepsin G inhibitory activity which are the objects of the present invention, does not show the trypsin inhibitory activity substantially, and is an antigen for humans. In the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which has the property of no activity or is substantially reduced,
The addition or deletion of one or more amino acids and / or
Alternatively, it refers to a substituted elastase inhibitory activity polypeptide or a homopolymer thereof.

As used herein, the phrase "does not substantially show trypsin inhibitory activity" means that trypsin inhibitory substances that would interfere with elastase inhibitory activity or cathepsin G inhibitory activity, which is the object of the present invention, would be an obstacle when used as a therapeutic drug for human diseases. It means that a physiological effect due to the activity is hardly generated. For example, the trypsin inhibitory activity is about 1/10 or less when compared with the trypsin inhibitory activity of natural SLPI in the measurement method of the example of the present invention. The trypsin inhibitory activity of the protein of the present invention is about 1 / about that of its elastase inhibitory activity.
It is 100 or less.

The homopolymer of the elastase-inhibiting activity polypeptide of the present invention means, for example, Cys in the amino acid sequence of a plurality of elastase-inhibiting activity polypeptides.
It refers to an oligomer or the like such as a dimer formed by a disulfide bond via a residue.

The elastase-inhibiting activity polypeptide or homopolymer thereof of the present invention has an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof having an elastase-inhibiting activity, or an amino acid sequence biologically equivalent thereto. Have. As is clear from the amino acid sequence represented by SEQ ID NO: 1, the polypeptide of the present invention is a Cys residue in which the Cys residue is not carboxymethylated, but the polypeptide of the present invention is preferably Examples thereof include those in which Cys residues mutually form a disulfide bond necessary for the inhibitory activity of the present invention. Among them, those having four disulfide bonds are preferable.

Hereinafter, the elastase-inhibiting activity polyamine of the present invention
Amino acid sequence of natural SLPI when referring to peptides
SEQ ID NO: 1 using the numbers from the N-terminus of
The lastase inhibitory polypeptide (Arg⁵⁸-Al
a¹⁰⁷) SLPI is sometimes called, but this (Arg
⁵⁸-Ala¹⁰⁷The amino acid sequence of) is the sequence listing of SEQ ID NO: 1.
In (Arg¹-Ala⁵⁰) Is supported. There
And, as an example having four disulfide bonds,
For example, like the disulfide bond in natural SLPI,
Cys⁶⁴-Cys⁹³, Cys⁹²-Cys⁸⁰, Cys⁷¹-
Cys⁹⁷, And Cys ⁸⁶-Cys¹⁰¹, That is, the sequence number
In the sequence listing of No. 1, Cys⁷-Cys³⁶, Cys³⁵
-Cys^{twenty three}, Cys¹⁴-Cys⁴⁰, And Cys²⁹-Cy
s⁴⁴Corresponding to the
Lipeptides can be mentioned as preferred.
However, the inhibitory activity and reduction aimed at by the present invention
At least 1 or more as long as the antigenicity is
The polypeptide or
It may be a mopolymer.

In addition, the elastase-inhibiting polypeptide represented by SEQ ID NO: 2 using the number from the N-terminal of the amino acid sequence of natural SLPI is (Arg ⁵⁹ -Ala ¹⁰⁷ ) SL.
Sometimes referred to as PI, this (Arg ⁵⁹ -Ala
The amino acid sequence of ¹⁰⁷ ) corresponds to (Arg ¹ -Ala ⁴⁹ ) in the sequence listing of SEQ ID NO: 1. Therefore, as those having four disulfide bonds, for example, Cys ⁶⁴ is similar to the disulfide bond in natural SLPI.
-Cys ⁹³ , Cys ^92- Cys ⁸⁰ , Cys ^71- Cy
s ⁹⁷ and Cys ⁸⁶ -Cys ¹⁰¹ , that is, Cys ⁶ -Cys ³⁵ , Cys ³⁴ -C in the sequence listing of SEQ ID NO: 2.
ys ²² , Cys ¹³ -Cys ³⁹ , and Cys ²⁸ -Cys ⁴³
A polypeptide having 4 disulfide bonds, corresponding to, can be mentioned as a preferable example. However, it may be a polypeptide or homopolymer having at least one disulfide bond as long as it has the inhibitory activity and reduced antigenicity which are the objects of the present invention.

The present invention further relates to the following formula [I] AX—B ... [I] which is useful as an intermediate for the production of the above-mentioned polypeptide, wherein A represents a carrier protein and B is a sequence. No. 1 or SEQ ID No. 2 or a part thereof having elastase inhibitory activity, or an elastase inhibitory polypeptide having an amino acid sequence biologically equivalent to them, or a part thereof having elastase inhibitory activity, or a homopolymer thereof X represents a cross-linked polypeptide having an amino acid sequence cleavable by a chemical method or a biological method under the condition that the target protein is not denatured). .

The present invention also provides a method for producing the above-mentioned elastase-inhibiting activity polypeptide or a homopolymer thereof, wherein the fusion protein represented by the above formula [I] is treated by a chemical method or a biological method. A method consisting of
And a method for producing the above-mentioned elastase-inhibiting activity polypeptide or a homopolymer thereof, which comprises chemically synthesizing the amino acid constituting the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. .

The present invention further provides a gene encoding a carrier protein and an elastase having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof having an elastase inhibitory activity, or an amino acid sequence biologically equivalent thereto. The gene encoding the inhibitory activity polypeptide is linked via a gene encoding a cross-linking peptide or a homopolymer thereof having a cleavable amino acid sequence by a chemical method or a biological method under the condition that the target protein is not denatured. And a fusion protein gene-expressing plasmid or recombinant microbial cell containing the fusion protein gene.

Furthermore, the present invention provides a pharmaceutical preparation comprising a therapeutically effective amount of the above-mentioned elastase inhibitory activity polypeptide or a homopolymer thereof and a pharmaceutically acceptable carrier, and a disease caused by neutrophil hyperactivation, Among them, there is provided a therapeutic agent for a disease caused by a neutrophil-releasing protease such as elastase and / or cathepsin G.

The fusion protein represented by the formula [I] of the present invention is a method for obtaining the fusion protein exemplified in WO 89/06239, for example, by a known means of gene recombination. A plasmid expressing the fusion protein is constructed using the fusion protein gene, the plasmid is introduced into a microbial host cell such as Escherichia coli, and the microbial cell is cultured, and then the transformed microbial cells are collected by, for example, centrifugation. It can be obtained by a method similar to the method for isolating a fusion protein from cells.

In the above formula [I], which represents a fusion protein useful as an intermediate for producing the elastase inhibitory activity polypeptide of the present invention, a carrier protein represented by A and X
Examples of the cross-linked polypeptide represented by
Mention may be made of those exemplified in the specification of 9/06239. The carrier protein represented by A has a high expression level of the fusion protein and is present as an inclusion body in microbial cells, so that it is easy to isolate and purify human growth hormone and a part thereof, or T7 phage gene10 protein (Studie).
r, F. et al, J. Mol. Biol. 189, 113, 198.
6) and parts thereof are preferably used. The human growth hormone also includes methionyl human growth hormone and modified forms of which the amino acid sequences are partially different from the natural ones. When a part of human growth hormone is used as a carrier protein, from Phe at the amino acid residue 1-position of human growth hormone to 139
Peptide fragments up to position Phe and polypeptide fragments from position 1 Phe to position 122 Gln are preferred.

The cross-linked polypeptide represented by X is an amino acid sequence which can be cleaved by a chemical method or a biological method under the condition that the target protein is not denatured, and is any recognized amino acid sequence including known ones. included.

The amino acid sequence which can be cleaved by a chemical method is, for example, Asn-Gly (P. Pornstein, Met as a cross-linking peptide cleavable by hydroxylamine.
h. Enzymol., 47 , 132, 1977), 2-1
There are 0 repeat sequences. An amino acid sequence cleavable by a biological method is, for example, an enzymatically cleavable amino acid sequence which is cleaved by thrombin (B.
Blomback et al., BBA 115 , 371-396, 196.
6; T. Takagi et al., Biochemistry 13 , 750-75.
6, 1974, etc.) amino acid sequence, especially Val-Pro-Arg or Leu-Val-Pr.
o-Arg, or an amino acid sequence cleaved by enterokinase, such as 2-10 repeating sequences thereof,
For example, Asp-Asp-Asp-Asp-Lys, or 2 to 10 repeating sequences thereof, such as those capable of reliably releasing and separating the target protein without being affected by the primary amino acid sequence of the target protein or the three-dimensional structure It can be used without particular limitation. A signal or the like can be appropriately added to these carrier proteins and cross-linked polypeptides, as required during gene expression.

The cross-linked polypeptide represented by X and the target protein comprising the elastase-inhibiting activity polypeptide represented by B or a homopolymer thereof are Arg at the amino terminus of the target protein and Gly, Arg and A of X, respectively.
It means that either rg or Lys is bound.

In order to produce the elastase-inhibiting activity polypeptide of the present invention, the fusion protein represented by the above formula [I] may be cleaved at the bridging amino acid or peptide X.

When such cleavage is carried out by enzymatic treatment, a method using an endopeptidase that recognizes a specific amino acid (single or plural) in the amino acid sequence of X and preferentially cleaves a specific peptide bond, etc. There is. As a method using endopeptidase, for example, thrombin or its homolog can be used, and examples of such thrombin or its homolog include human thrombin, bovine thrombin, horse thrombin, pig thrombin and the like. In this case, X as Val-Pro-
It is preferred to use Arg, Leu-Val-Pro-Arg, or repetitive sequences thereof. It is also possible to use enterokinase or its homologue, and examples of such enterokinase or its homologue include bovine enterokinase (VN Dobrynin et al., Bioory-Khim) for releasing enkephalin from β-galactosidase.
13 , 119-121, 1987), enterokinase for releasing human atrial natriuretic factor from chloramphenicol acetyltransferase (A. Hobden et al., WPI 87-0888509 / 13) and the like. In this case, X is, for example, A
It is preferred to use sp-Asp-Asp-Asp-Lys, or repetitive sequences thereof.

When the cleavage is carried out by a chemical treatment, for example, hydroxylamine or its homologue can be used. Examples of the hydroxylamine or its homologue include alkylhydroxylamine, hydrazine and the like. . Asn in this case is preferably Asn-Gly, or a repeating sequence thereof.

The fusion protein represented by the formula [I] of the present invention can be produced by a known method of gene recombination. In this case, in the present invention, a gene encoding a carrier protein consisting of human growth hormone or a fragment thereof and a gene encoding the target protein or a part thereof are treated by a chemical method or under a condition that the target protein is not denatured. It is characterized by using a fusion protein gene linked via a gene encoding a peptide or polypeptide having an amino acid sequence cleavable by a biological method.

A plasmid (pGH) expressing a fusion protein in which a (Met ^-1 Phe ¹ -Phe ¹³⁹ ) human growth hormone fragment and an (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI fragment polypeptide are linked via a thrombin cleavage sequence. -T5
The method for constructing 9) is shown in FIG.

A plasmid expressing a fusion protein in which a (Met ^-1 Phe ¹ -Phe ¹³⁹ ) human growth hormone fragment and an (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI fragment polypeptide are linked via an enterokinase cleavage sequence ( pGH
A method for constructing —E59) is shown in FIG.

A plasmid (pGH-) expressing a fusion protein in which the (Met ^-1 Phe ¹ -Phe ¹³⁹ ) human growth hormone fragment and the (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI fragment polypeptide are linked via a thrombin cleavage sequence. T5
The method for constructing 8) is shown in FIG.

The (Met ^-1 Phe ^-1 -Phe ¹³⁹ ) human growth hormone fragment and the (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI fragment polypeptide were combined (Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp).
-Lys) using a fusion protein gene expression type plasmid (pGH-E59) containing a fusion protein expression gene linked via an enterokinase cleavage sequence gene as a template DNA,
Amplification was repeated by polymerase chain reaction using the synthetic DNA fragment (shown in FIG. 14) and the synthetic DNA fragment (shown in FIG. 14) as primers, and the resulting amplification product was cleaved with BglII to form a human growth hormone fragment. -Crosslinked polypeptide- (Arg ⁵⁸
-Ala ¹⁰⁷ ) Obtain a gene fragment consisting of SLPI fragment.

On the other hand, another pGH-E59 is cleaved with BglII to obtain a BglII-BglII-cleaved gene fragment containing the Trp promoter, and this fragment is dephosphorylated at the 5'end. The two fragments thus obtained are ligated to obtain a fusion protein expression type plasmid pGH-T58 of the human growth hormone fragment and the (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI fragment. In FIG. 14, p
GH-T58 is a fusion protein expression plasmid having a thrombin cleavage sequence.

In addition, as in the method of constructing pGH-T59, human growth hormone expression plasmid pGH-L9
(Proc. Natl. Acad. Sci. USA., 81 , 5956, 19
84) A gene fragment obtained by removing the latter half of the human growth hormone gene and SLPI subclone pUC-D
6 (WO89 / 06239) was cleaved with a restriction enzyme to obtain (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI fragment, and ligated with a synthetic DNA linker (for example, a DNA fragment containing a thrombin cleavage sequence gene). An expression type plasmid pGH-T58 can also be obtained in the same manner as in.

In the present invention, the gene encoding the recognition amino acid sequence of the fusion portion linking with the gene encoding human growth hormone or a part thereof has the same reading frame as the carrier protein human growth hormone or a part of the gene thereof. There is no particular limitation as long as it is an amino acid sequence that can be easily cleaved by treating the expressed fusion protein with the aforementioned chemical method or biological method to separate the target protein. For example, (Asn-Gly) _n , (Asp-A
sp-Asp-Asp-Lys) _n , (Leu-Val
-Pro-Arg) _n , (Val-Pro-Arg) _n
Any gene may be used as long as it encodes (n represents an integer of 1 to 10).

A synthetic DNA linker may be inserted between the carrier protein and the genes encoding these recognition amino acid sequences in order to align the reading frame.

Also, (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI
As a gene fragment, this (Arg ⁵⁸ -Ala ¹⁰⁷ ) SL
A gene corresponding to a PI polypeptide fragment, or 2 to
It is also possible to use 10 repetitive genes.

The fusion protein gene of the present invention can be made into an expressed gene by connecting it to the downstream of an appropriate promoter and SD sequence. As usable promoters, tryptophan operon promoter (trp promoter), lactose operon promoter (la)
c promoter), tac promoter, PL promoter,
Examples include Ipp promoter, T7 promoter, and the like. Particularly, the 5'flanking sequence of pGH-L9, which uses the trp promoter and optimizes the interval between the SD sequence and the translation initiation signal ATG, is preferable (JP-A-60-234584).
Issue).

A fusion protein gene-expressing DNA for efficiently expressing a fusion protein includes a trp promoter, an SD sequence, a translation initiation codon, a gene encoding human growth hormone or a part thereof, and a gene encoding a cross-linking peptide. , And a gene encoding the protein of interest or a part thereof, and further a translation termination codon linked in this order. Such fusion protein gene expression type DNA
Can be inserted into an appropriate plasmid, such as pBR322 and a plasmid related thereto, to prepare a fusion protein gene expression type plasmid. A preferred plasmid is pBR322.

Microbial host cells for expressing the fusion protein gene of the present invention include prokaryotic cells such as Escherichia coli, Bacillus subtilis, actinomycetes; mammalian cells such as CHO cells and COS cells; insects such as silkworms. The cells include eukaryotic cells such as yeast cells. E. coli cells are particularly preferable.

The above fusion protein gene expression type plasmid is
For example, known methods (MV Norgard et al., Gene, 3 , 27.
9, 1978) can be used to introduce microbial host cells such as E. coli cells.

The thus-obtained transformed microorganism can be obtained by a method known per se, for example, WO89 / 06.
No. 239, the method of culturing under the kind of medium and culturing conditions, collecting the transformed microbial cells after culturing, crushing this, and isolating and purifying the fusion protein to obtain the fusion protein. be able to.

The obtained fusion protein can be cleaved by treatment with the above-mentioned chemical method or biological method, and if necessary, it is sulfonated and treated as a sulfonated molecule to separate the intact target protein.・ Can be released.

In the method for producing the elastase inhibitory activity polypeptide or the polymer thereof of the present invention, human leukocyte elastase inhibitory protein (SLPI) is treated by an enzymatic method or the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The method consisting of chemically synthesizing using the amino acids constituting the can be carried out by the following method.

The SLPI in the method for enzymatically treating SLPI of the present invention is a human leukocyte elastase inhibitor protein which is present in human saliva, nasal discharge, semen, uterine mucus, etc., such as parotid secretion. Origin S
LPI (RC Thompson et al., Proc. Natl. Acad. Sci. U
SA, 83 , 6692-6696, 1986; Tokusho Sho 62
-501291), natural human SLPI; recombinant natural human SLPI obtained by genetic engineering using a gene encoding the amino acid sequence of natural human SLPI; or WO 89/06239. Recombinant natural type (Asn ⁵⁵ -Ala ¹⁰⁷ ) human SLPI fragment obtained by the described genetic engineering method can be mentioned.

Examples of the enzyme used for the enzyme treatment include the enzymes present in human saliva, nasal discharge, semen, uterine mucus, etc., such as human sputum-derived polymorphonuclear leukocyte elastase, which include these enzymes. Human saliva or the like can also be used.

As the treatment conditions in such an enzymatic method, it is preferable to act at about 20 to 45 ° C. for 30 minutes to 72 hours.

In the method for producing the elastase-inhibiting activity polypeptide of the present invention or a homopolymer thereof, a method of chemically synthesizing the amino acid constituting the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is as follows: For example, it can be synthesized using such an amino acid, which is a conventionally known method, as a raw material using an automatic peptide synthesizer.

The elastase-inhibiting activity polypeptide or the homopolymer thereof, which is the target protein thus obtained, can be separated and purified from the reaction solution by an appropriate combination of separation and purification methods known per se. Known separation and purification methods of these include those utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Difference-based methods, ion-exchange chromatography, ion-exchange high-performance liquid chromatography, and other charge-difference methods, affinity chromatography, and specific affinity methods, reverse-phase high-performance liquid chromatography, and other hydrophobic methods Examples thereof include a method of utilizing a difference in sex and a method of utilizing a difference in isoelectric point such as an isoelectric focusing method. When the obtained target protein or the like does not have a higher-order structure having biological activity,
The target protein can also be converted into a higher-order structure protein having biological activity by using a disulfide bond forming reaction known per se.

When the target protein thus obtained has a higher-order structure through a disulfide bond, for example,
Chance et al., Peptides: 7th American Peptide Symposium Proceedings (DH Rich and
E. Gross), 721-728, Pierce Chemical Co.,
Rockford, IL. (1981)) or the method disclosed in Japanese Patent Application No. 2-141523 previously disclosed by the present inventors, whereby a biological structure having a higher-order structure similar to that of a natural protein can be obtained. Can be a highly active protein.
In the thrombin cleavage method, which involves a sulfonation method, the sulfonation target protein is formed into a biologically active protein having the same higher-order structure as the natural protein by forming an intramolecular disulfide bond directly or after reduction. You can

The elastase-inhibiting activity polypeptide of the present invention has the property of exhibiting elastase-inhibiting activity and cathepsin G-inhibiting activity, and substantially no trypsin-inhibiting activity, and since it is derived from a natural type, it is an antigen. Since it is expected that there is no activity or that antigenicity is substantially reduced, various diseases, for example, diseases caused by neutrophil hyperactivation, among them, for example, elastase and / or cathepsin G, etc. , A disease caused by a neutrophil-releasing protease, for example, a therapeutic agent for inflammatory disease, platelet aggregation thrombosis, or post-ischemic reperfusion injury, particularly a respiratory disease among inflammatory diseases, such as chronic bronchitis, Diffuse panbronchiolitis, emphysema, bronchiectasis, bacterial pneumonia, sinusitis, A
It can be used as a therapeutic drug for RDS (respiratory distress syndrome), asthma, interstitial pneumonia, and the like.

When the elastase-inhibiting activity polypeptide of the present invention is used as a pharmaceutical preparation or a therapeutic agent, it can be lyophilized into a powder, if necessary, and in that case, for example, sorbitol, mannitol, dextrose, Stabilizers such as maltose, glycerol and human serum albumin can also be added.

The pharmaceutical preparation and therapeutic agent of the present invention can be administered through the respiratory tract or orally, or parenterally such as intravenously, subcutaneously, intramuscularly, transdermally, and rectally.

As a dosage form for administration via the respiratory tract, an inhalation preparation or a spray-form preparation comprising a liquid preparation and a powder preparation is known. If necessary, a bacteriostatic agent and an antioxidant can be added as additives.

Examples of the dosage form for oral administration include tablets, pills, granules, powders, suspensions and capsules.

To form tablets, for example, excipients such as lactose, starch and crystalline cellulose; binders such as carboxymethyl cellulose, gelatin and polyvinylpyrrolidone; disintegrators such as sodium alginate, starch and crystalline cellulose are used. And can be molded by a usual method.

Pills, powders and granules can also be formed by the usual methods using the above-mentioned excipients and the like.

The liquid agent and the suspension agent can be formed by a usual method using, for example, glycerin esters such as triacetin, alcohols such as ethanol and the like. Capsules can be formed by filling granules, powders or liquids into capsules such as gelatin.

As a dosage form for subcutaneous, intramuscular or intravenous administration, an injection in the form of an aqueous or non-aqueous solution is known. As the aqueous solution, physiological saline or the like is used. As the non-aqueous solution, for example, propylene glycol, polyethylene glycol, olive oil, ethyl oleate, etc. are used, and if necessary, a preservative,
Stabilizers and the like are added. The injection is sterilized by appropriately performing treatments such as filtration through a bacteria removal filter and addition of a germicide.

The dosage form for transdermal administration is, for example, an ointment,
Creams and the like can be mentioned. Ointments are made by a conventional method using fatty oils such as castor oil and olive oil; petroleum jelly and the like, creams are fatty oils; emulsifiers such as diethylene glycol and sorbitan monofatty acid ester. be able to.

For rectal administration, usual suppositories such as gelatin soft capsules are used.

The dose of the polypeptide of the present invention varies depending on the route of administration, the age of the patient, the sex, and the severity of the disease.
It is usually about 1 to 5000 mg / dose, and the number of doses is usually 1
It is ~ 3 times / day, and it is preferable to adjust the formulation so as to satisfy such conditions.

[0073]

EXAMPLES The present invention will be described in more detail with reference to reference examples and examples, but it goes without saying that the present invention is not limited to these examples.

Various genetic engineering techniques adopted in Reference Examples and Examples were carried out by the methods described in WO 89/06239, pages 24 to 26.

[0075]

Reference Example 1 Production of (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI According to the example of WO 89/06239 (As
n ⁵⁵ -Ala ¹⁰⁷ ) SLPI was prepared.

That is, (Asn ⁵⁵ -Ala ¹⁰⁷ ) SL
A DNA fragment encoding PI was chemically synthesized using appropriate codons. Leu that can be cleaved with thrombin
The human growth hormone gene (Asn ⁵⁵ -Al is inserted through the DNA sequence encoding Val-Pro-Arg.
a ¹⁰⁷ ) fused to SLPI. The expression vector thus obtained (plasmid pGH-TE) was transformed into E. col
It was introduced into iHB 101. The transformed cells (Escherichia coli HB 101 strain (pGH-TE)) were cultured to obtain a complex, which was purified by thrombin treatment, SS rearrangement and chromatography to obtain the desired (Asn ⁵⁵ -Al).
a ¹⁰⁷ ) SLPI was obtained. Obtained (Asn ⁵⁵ -Ala
¹⁰⁷ ) SLPI is Cys ⁶⁴ -Cys ⁹³ , Cys ⁹²
-Cys ⁸⁰ , Cys ^71- Cys ⁹⁷ , and Cys ^86- Cy
It was confirmed by Mass spectrum and two-dimensional NMR that the Cys residues of s ¹⁰¹ were forming four disulfide bonds with each other.

The Escherichia coli HB 101 strain (pGH
-TE is Micro Engineering Research Institute No. 2168 (FERM BP-
2168), 19
It has been internationally deposited on December 1, 1988.

[0078]

Reference Example 2 Production of SLPI Amino acid sequence of SLPI (Proc. Natl. Acad. Sci. US
A, 83, 6692-6696, 1986) was chemically synthesized. WO89 / 06
In the same manner as in the example of US Pat. No. 239, SL was added to the human growth hormone gene via a DNA sequence encoding Leu-Val-Pro-Arg which can be cleaved by thrombin.
The DNA encoding PI was fused. The expression vector thus obtained (plasmid pGH-SLPI) was transformed into E. E. coli HB 101. Transformed cells (Escherichia coli HB 101 strain (pGH-SLPI))
C. to obtain a complex, which is purified by thrombin treatment, SS rearrangement and chromatography to obtain the desired S
Got the LPI. The inhibition constant (Ki) of the obtained SLPI for elastase was 2.5 × 10 ^-10 M, which was equivalent to the activity of SLPI isolated from nature (Table 62-5).
No. 01291).

[0079]

Example 1 Identification of SLPI Degradation Products by Normal Human Saliva (1) After normal filtration of normal human saliva with a 0.22 μm filter (Millex GS, Millipore), the SLPI obtained in Reference Example 2 was obtained. It was dissolved in a concentration of 1 mg / ml and kept at 37 ° C for 30 days. 1 mg / ml of saliva after filtration and SLPI in physiological saline (Fishartz, Fuso Yakuhin Kogyo)
What was dissolved in the concentration of
The temperature was kept at 30 ° C for 30 days.

After collecting each of these samples over time, Tr
is-HCl buffer (pH 6.8), SDS, 2-
Mercaptoethanol and glycerol were added to final concentrations of 60 mM, 2 w / w%, 4 w / w%, and 10 w / w%, respectively, and SDS-PAGE (UK Laemmli, Nature,
227 , 680, 1970). The result is shown in Figure 1.
Shown in. The gel after electrophoresis was stained with Coomassie Brilliant Blue. Lane No. 2 band a (molecular weight about 13,000) shows the band of SLPI.

As is clear from FIG. 1, the first temperature retention at 37 ° C.
After the day, the molecular weight was about 10,000 (band b),
After the third day, new bands appeared at a molecular weight of about 7,000 (band c) and a molecular weight of about 3,000 (band e). Furthermore, a molecular weight of about 13,000 is conceived for SLPI.
It was confirmed that the band a of No. 1 decreased as the heat retention time was extended, while the bands b, c and e increased. In addition, SLPI in physiological saline solution is
A band with a molecular weight of about 13,000 was maintained (lane N
o. 9). In addition, SDS-P after 30 days of incubation at 37 ° C
From the result of AGE, band d having a molecular weight of about 5,500 was confirmed.

(2) Next, the polypeptide in the polyacrylamide gel thus obtained was treated with Milli Blot-SDE.
Using a device (Millipore), the membrane (Im
mobilon, Millipore). After the transfer, the membrane was stained with 0.1 w / w% Coomassie Seel / 40 w / w% methanol / 1 w / w% acetic acid and washed with water. After air drying, bands b, c, d, and e were cut out, and Applied Biosystems protein sequencer (manufactured by Applied Biosystems; 47).
0A), PTH-amino acid is cleaved from the N-terminus, and a PTH analyzer (manufactured by Applied Biosystems, 1
20A) and the amino acid sequence from the N-terminal to the 10th longest was determined. The results are shown in Table 1. In addition,
There were two N-terminal amino acid sequences of band e.

[0083]

[Table 1]

From Table 1, bands b and c are SLPI Ty.
The amino acid sequence from r ²¹ , the band d, the amino acid sequence from Arg ⁵⁹ , and the band e completely matched the SLPI amino acid sequence from Leu ⁷⁴ or Met ⁷³ . Therefore, the selective cleavage site of SLPI in normal human saliva is Arg.
^{Between 20-} Try ²¹ , between Arg ^{58 and} Arg ⁵⁹ , Leu ⁷²
During the -Met ^73, it was confirmed that between the Met ⁷³ -Leu ^74.

From the N-terminal amino acid sequences of bands b, c, d and e (Table 1) and the molecular weights (FIG. 1), it was confirmed that the polypeptides were the polypeptides shown in Table 2 below. It was

[0086]

[Table 2]

[0087]

[Example 2] (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI by normal human saliva
Degradation product identification (1) By the same method as in Example 1, (Asn ⁵⁵ -Ala)
¹⁰⁷ ) The results of SDS-PAGE when SLPI was saliva treated and each sample was collected over time are shown in FIG. When the (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI (molecular weight of about 5,800, band f) obtained in Reference Example 1 was kept warm at 37 ° C in normal human saliva, the molecular weight of about 5,500 was obtained from the 12th hour. Band g newly appeared. The band f gradually disappeared as the heat retention time was extended, and only the band g was left after 96 hours. In addition, (Asn ⁵⁵ -Al
a ¹⁰⁷ ) SLPI was a single band f having a molecular weight of about 5,800 even after 96 hours at 37 ° C.

(2) Next, this band g was electrically transferred to a membrane in the same manner as in Example 1, and then the N-terminal amino acid sequence was analyzed. The results are shown in Table 3.

[0089]

[Table 3]

From this result, the N-terminal amino acid sequence of band g was the SLPI amino acid sequence after Arg ⁵⁹ .

From the above, (Asn ⁵⁵ -Ala ¹⁰⁷ ) SL
PI is cleaved between Arg ^{58 and} Arg ⁵⁹ in normal human saliva, and all (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLP
It was confirmed that it could be converted to I.

[0092]

Example 3 Polypeptide of the present invention: (Arg ⁵⁹ -Ala ¹⁰⁷ ) SL
Production of PI The polypeptide of the present invention represented by SEQ ID NO: 2 was converted into an automatic peptide synthesizer (manufactured by Applied Biosystems, 4
31A) and reverse phase chromatography (YM
C-Pack ODS-AP, YMC). Since the obtained peptide has an unnatural intramolecular and intermolecular disulfide bond, (Arg ⁵⁹ -Ala ¹⁰⁷ ) SL
After converting to a PI sulfonated derivative and cleaving all disulfide bonds, it has a natural type intramolecular disulfide bond by a refolding reaction (Arg ⁵⁹ -Ala).
¹⁰⁷ ) Got SLPI. That is, 20 mg of the polypeptide of SEQ ID NO: 2 synthesized by an automatic peptide synthesizer was
25M Tris Buffer (pH 8.2), 3.
After dissolving in 20 ml of 5M urea, 0.35M Na ₂ SO ₃ and 0.06M sodium tetrathionate and reacting at room temperature for 2 hours, the reaction solution was subjected to reverse phase chromatography (Prot.
Ein C ₄ , VYDAC) separated and purified, and then freeze-dried (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI sulfonated derivative 18
to obtain mg.

The refolding reaction and purification were carried out by the same method as that described in Japanese Patent Application No. 2-141523.

10 mg of this sulfonated derivative was added to 0.1 M T
ris Buffer (pH 8.5), 0.2 mM oxidized glutathione, 1.5 mM reduced glutathione 50 ml
And was reacted at 4 ° C. for 7 days. Then S-Seph
arose (Pharmacia) ion exchange chromatography, reverse phase chromatography (Protein C ₄ , VYD)
AC company), and the oligomer by-produced by reholding was separated. Then lyophilize and (Arg ⁵⁹ -A
la ¹⁰⁷ ) SLPI 7.8 mg was obtained. The obtained polypeptide was subjected to reverse phase chromatography (Protein C ₄ , VY
When analyzed by DAC), it was eluted as a single peak and confirmed to be a single substance. The elution pattern is shown in FIG.

[0095]

Example 4 (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI elastase and
The measurement method of the trypsin inhibitory activity is shown below.

(1) Reagent solution buffer solution : 0.1 M N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES),
1.0M NaCl, 0.1% PEG6000 (pH
7.5) Enzyme solution : Each of the following enzymes is dissolved in the above buffer solution so as to have a concentration 9 times the final concentration shown in Table 4. (A) Human sputum-derived polymorphonuclear leukocyte elastase (EPC
(Funakoshi Pharmaceutical Co., Ltd.) (b) Bovine spleen trypsin (manufactured by Sigma) Substrate solution ; Substrate (ha) below for the above enzyme (a),
For (b), the substrate of (d) was adjusted to 9 times the final concentration shown in Table 4, so that (c) was DMSO; the above buffer was 1: 9, and (d) was DMSO only. Dissolve into a substrate solution. (C) MeO-Suc-Ala-Ala-Pro-Va
l-pNA (manufactured by Bachem) (d) Bz-Arg-pNA (manufactured by Bachem)

[0097]

[Table 4]

(2) Method 120 μl was added to the well of the 96-well microplate for ELISA.
Buffer, then 20 μl of sample solution of (Arg ⁵⁹ —Ala ¹⁰⁷ ) SLPI obtained in Example 3 was added, followed by 20 μl of enzyme solution and the mixture was added to 3
The mixture was stirred at 7 ° C for 1 hour. Next, 20 μl of the substrate solution was added, and the mixture was stirred at 37 ° C. for 15 minutes to develop color, and the absorbance was measured at 405 nm. SLPI was used as a control, the concentration of the solution was changed, the inhibitory activity was measured, and the inhibitory protein concentration (IC ₅₀ value) showing an inhibitory concentration of 50% was determined. The results are shown in Table 5.

The IC ₅₀ value of the oligomer by-produced in Example 3 for elastase measured by the same method was 3.0 × 10 ^-7 M.

[0100]

[Table 5]

[0101]

Example 5 Inhibition of (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI by Cathepsin G
Activity measurement Cathepsin G inhibitory activity was measured in the same manner as in Example 4. 120 in the hole of 96-well microplate for ELISA
μl buffer (0.1M HEPES, 1.0M Na
Cl, 0.1% PEG6000 pH 7.5),
Then 20 μl of the product obtained in Example 3 (Arg ⁵⁹ -Ala) was used.
¹⁰⁷ ) SLPI sample solution was added, followed by 20 μl of cathepsin G solution (final concentration 1.1 × 10 ⁻⁸ M) and the mixture was stirred at 37 ° C. for 1 hour. Then 20μ
l substrate solution (Suc-Phe-Pro-Phe-pN
(A; final concentration 1.1 mM) was added, and the mixture was stirred at 37 ° C. for 2 hours to develop color, and the absorbance was measured at 405 nm. By changing the concentration of the sample solution and measuring the inhibitory activity, 5
Inhibition shows the inhibitory concentration of 0% protein concentration (IC ₅₀ value) result of obtaining the value of (Arg ⁵⁹ -Ala ¹⁰⁷⁾ SLPI an IC ₅₀ is 1.1 × 10 ^-8 M, strong against cathepsin G It exhibited inhibitory activity.

[0102]

Example 6 Human growth hormone fragment polypeptide and (Arg ⁵⁹ -Al
a ¹⁰⁷ ) SLPI polypeptide fusion protein expression plasmid
Preparation of Building De and its transformant (1) expression plasmid crosslinked peptide has thrombin cleavage sequence (Met ^-1 Phe ¹ -Phe ¹³⁹⁾ human growth hormone expression plasmid pGH-L9 (M. Ikehara et, Proc. Nat
l. Acad. Sci. USA, 81 , 5956, 1984) was cleaved with restriction enzymes BglII and SalI (Takara Shuzo), separated by agarose electrophoresis, and a DNA of about 4.7 kbp was obtained from the gel.
The fragment was recovered using GENECLEAN II KIT (manufactured by BIO 101).

Also, the SLPI subclone pUC-D6
(WO89 / 06239) with restriction enzymes MluI and Xho
Cleavage with I (Takara Shuzo), separation by agarose electrophoresis, and DNA fragment of about 0.15 kbp from MERmaid
It was recovered using ^TM kit (manufactured by BIO101).

On the other hand, D containing a thrombin cleavage sequence gene
The NA fragment (FIG. 4) was synthesized by a fully automatic synthesizer (Applied Biosystems model 392). Synthetic DNA fragment is MEGALABEL
After phosphorylating the 5'end with ^TH (Takara Shuzo) in the presence of 1 mM ATP, it was annealed with a synthetic DNA fragment to obtain a double-stranded DNA. The annealing reaction was heated at 90 ° C. for 5 minutes and then allowed to reach room temperature.

The construction of the fusion protein expression plasmid begins with pGH-L9 BglII-SalI approximately 4.7 kbp.
Double-stranded synthetic DN containing DNA fragment and thrombin cleavage site
After performing a ligation reaction using A with a ligation kit (Takara Shuzo) at a molar ratio of 1:10, A-free C3H
It was concentrated with a KUF filter (MILLIPORE). After further ligation reaction with about 0.15 kbp DNA fragment of pUC-D6 MluI-XhoI, E. coli HB101 competent cell (Takara Shuzo)
And cultured overnight on LB agar medium containing ampicillin to obtain a transformant (HB / GH-T59) containing the desired fusion protein expression plasmid. Extraction of plasmid DNA from the transformant, QIAGEN (manufactured by BIO101)
Purified with a human growth hormone cross-section and (Arg ⁵⁹ -Ala
¹⁰⁷ ) A fusion protein expression plasmid pGH-T59 of SLPI fragment polypeptide was obtained (FIG. 5). The DNA base sequence is Dye Terminator k using plasmid DNA as a template.
Using it (Hitachi), it was directly confirmed using Hitachi fluorescent automatic DNA sequencer SQ3000.

(2) Synthetic DNA using the expression plasmid pGH-T59 (FIG. 5) in which the cross-linking peptide has an enterokinase cleavage sequence as the template DNA
Polymerase chain reaction using the fragment (FIG. 6) as a primer; PCR method (Saiki et al., Science, 23
9 , 487-491, 1985), the amplified product was again amplified by the PCR method using the synthetic DNA fragment, (FIG. 6) as a primer, and then digested with BglII (Takara Shuzo) to obtain a fragment of about 115 bp. Obtained.

The PCR conditions were such that denaturation was carried out at 93 ° C. for 5 minutes, and then 1 cycle was performed at 93 ° C. for 1.5 minutes.
30 cycles of ℃, 2 minutes, 72 ℃, 2 minutes,
Finally, the reaction was stopped at 72 ° C for 7 minutes.

On the other hand, after cutting pGH-T59 with BglII (Takara Shuzo), about 4.6 kbp BglII-BglII fragment was separated by agarose gel electrophoresis.
It was recovered using EAN II (manufactured by BIO 101). The BglII-BglII fragment of about 4.6 kbp was dephosphorylated at the 5'end using Bacterial Alkaline Phosphatase (Takara Shuzo) according to the Takara Shuzo protocol.

Dephosphorylated approximately 4.6 kbp BglII
-BglII fragment and about 115 bp amplified by PCR method
The BglII-BglII fragment of E. coli was subjected to a ligation reaction using a ligation kit (Takara Shuzo), and then E. coli HB1
01 competent cells (Takara Shuzo) and cultured on LB agar medium containing ampicillin to obtain a transformant (HB / GH-E59) containing the desired fusion protein expression plasmid.

Plasmid DNA was extracted from the transformant and purified by QIAGEN (manufactured by BIO101) to have a human growth hormone fragment polypeptide and enterokinase fragment sequence gene (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI.
Polypeptide fusion protein expression plasmid pGH-E59
Was obtained (FIG. 7).

The nucleotide sequence was directly confirmed by a fluorescent automatic DNA sequencer SQ3000 manufactured by Hitachi using Dye Terminator kit (Hitachi) using plasmid DNA as a template.

[0112]

EXAMPLE 7 Fusion obtained in Expression Example 6 of the fusion protein gene protein gene expression plasmid pG
E. coli HB101 strain containing H-T59 was added to 0.25
% Glucose, 3 mg / ml yeast extract (manufactured by DIFCO) Modified M9 medium (1% (NH ₄ ) ₂ HPO ₄ -0.3%) containing 20 mg / ml casamino acid (manufactured by DIFCO)
K ₂ SO ₄ -0.3% NaCl) after autoclaving and then adding MgSO ₄ and CaCl ₂ to a final concentration of 1
It was inoculated into 500 ml of a culture solution aseptically added so as to be mM, and cultured overnight.

This overnight culture solution was used as the same culture solution 1 as described above.
To 0 liter, OD660 was added to be 0.2, and jar fermenter culture was performed at 37 ° C. O
When D660 reached 20, 3-β-indoleacrylic acid was added to the culture medium so that the final concentration was 150 μg / ml, and the culture was further continued at 37 ° C. for 4 hours. After that, E. coli cells were collected by centrifugation and washed with 0.5 M Tris buffer (pH 8.0).

The washed cells were suspended in 0.5 M Tris buffer containing lysozyme (1/200 of wet cell weight) and EDTA.3Na (1/20 of wet cell weight) and sonicated. Generator (BRANSON, CELL DISRU
After disrupting the cells using PTOR 900), centrifuge to separate the desired fusion protein into the precipitate (inclusion body).
obtained as y).

E. coli H containing pGH-T59
The B101 strain (HB / GH-T59) is 38
No. 63 (FERM BP-3863) has been deposited internationally on May 20, 1992 at the Institute of Microbiology, Institute of Industrial Science.

E. coli H containing pGH-E59
B101 strain (HB / GH-E59)
No. 61 (FERM BP-3861) has been deposited internationally on May 20, 1992 at the Institute for Microbial Industry, Institute of Industrial Technology.

[0117]

Example 8Sulfonation of Cysteine Residues of Fusion Proteins and Sulfonated Fusion Proteins
Cleavage reaction with white thrombin 25 g of the fusion protein obtained in Example 7 was added to 0.5 M Tris.
After suspending in 200 ml of buffer (pH 8.0), urine
240 g of element was added to solubilize it, and the final concentration was 0.7M.
Sodium sulfite (Wako Pure Chemical Industries, Ltd.)
The reaction was carried out at 5 ° C for 20 minutes. Furthermore, the final concentration should be 0.1M.
Sodium 4-thionate (Sigma)
And reacted at 45 ° C. for 20 minutes. 10 mM Tris-Ba
To the reaction solution (1 liter).
After that, ultrafiltration (rich
Repeat the operation of performing the filter (FILTRON) five times.
I returned. In this way, the sulfonated fusion protein solution obtained was obtained.
300 for liquid (the part that did not pass through the ultrafiltration membrane)
Add 0U of Usis Thrombin (Mochida Pharmaceutical Co., Ltd.) and add 3 at 37 ℃.
Reacted for hours. Then, 300K filter by ultrafiltration
Included in the solution that passed through the filter (Arg⁵⁹-Ala
¹⁰⁷) The SLPI sulfonated derivative was subjected to reverse phase HPLC (VY
DAC Company; Protein C_Four) And freeze-dried. Profit
(Arg ⁵⁹-Ala¹⁰⁷) SLPI sulfonation induction
N-terminal amino acid sequence of the body, protein sequencer
(Applied Biosystems 477A).

[0118]

Example 9 Activity of (Arg ⁵⁹ —Ala ¹⁰⁷ ) SLPI Sulfated Derivative
Refolding (disulfide formation reaction) into a functional molecule (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI obtained in Example 8
1 g of the sulfonated derivative was added to 5 liters of Refolding buffer (0.05 M Tris-HCl, pH 8.5, 0.3).
mM oxidized glutathione, 1.5 mM reduced glutathione), and reacted at 4 ° C. for 2 days. Then, the reaction solution was subjected to S-Sepharose (Pharmacia) ion exchange chromatography, and the elution pattern is shown in FIG. Show.
Fractions of interest eluting with 0.3 M NaCl were applied to reverse phase HP.
It was further purified by LC (VYDAC, Protein C ₄ ) and then lyophilized to give the active form (Arg ⁵⁹ -Ala ¹⁰⁷ ) SL.
I got PI.

[0119]

Example 10 (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI Serine Proteare
Measurement of ze inhibitory activity (1) The measuring method is shown below.

(1) Reagent solution Buffer solution: 0.1 M- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES) 0.5
M NaCl, pH 7.5 Enzyme concentration: Each of the following enzymes is dissolved in the above buffer solution to a concentration 9 times that shown in Table 6. A. Human sputum-derived polymorphonuclear leukocyte elastase (EPC, Funakoshi Pharmaceutical) B. Bovine spleen trypsin (manufactured by Sigma) C.I. Bovine spleen chymotrypsin (manufactured by Sigma) D. Pig spleen elastase (manufactured by Sigma) E. Human-derived cathepsin G (manufactured by EPC, Funakoshi Pharmaceutical) Substrate solution: Enzyme A. B. C. D. Each of the following substrates a, b, c, d, and e for each E was added to DMSO: buffer solution (1:
9) Dissolve in solution (DMSO final concentration 10 in buffer)
%) Substrate solution. a. MeO-Suc-Ala-Ala-Pro-Val
-PNA (manufactured by Bachem) b. Bz-Arg-pNA (manufactured by Bachem) c. Suc-Ala-Ala-Pro-Phe-pNA
(Manufactured by Bachem) d. Suc-Ala-Ala-Ala-pNA (Hanai Chemical) e. Suc-Phe-Pro-Phe-pNA (Bac
made by hem)

[0121]

[Table 6]

(2) Method 120 μl was added to the well of the 96-well microplate for ELISA.
Buffer, and then 20 μl of sample solution of (Arg ⁵⁹ —Ala ¹⁰⁷ ) SLPI obtained in Example 9 was added, followed by 20 μl of enzyme solution, and the mixture was added to 3 times.
The reaction was carried out at 7 ° C for 10 minutes. Next, 20 μl of the substrate solution was added, color was developed at 37 ° C., and the absorbance was measured at 405 nm.

SLPI was used as a control, the concentration of the solution was changed, the inhibitory activity was measured, and the inhibitory protein concentration (IC ₅₀ value) showing an inhibitory concentration of 50% was determined. The results are shown in Table 7.

[0124]

[Table 7]

[0125]

Example 11 (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI serine protector
(2) In the four enzymes shown below, (Arg ⁵⁹ -Ala)
¹⁰⁷ ) The inhibitory protein concentration (IC ₅₀ ) value showing 50% inhibition of SLPI and SLPI as a control was determined. F. Human plasma-derived thrombin (manufactured by Sigma) G. Human plasma-derived plasmin (manufactured by Sigma) Human plasma-derived kallikrein (Funakoshi Pharmaceutical, manufactured by Protogen) I. The pig-derived FXa (Daiichi Pure Chemicals) measurement method is shown below.

(1) Reagent Solution Buffer Solution: The buffer solution for each enzyme is shown below.

Thrombin, FXa; 50 mM Tris
-HCl, 7.5 mM EDTA-2Na, 175 mM
NaCl, pH 8.4 plasmin; 50 mM Tris-HCl, 100 mM
NaCl, pH 7.4 kallikrein; 50 mM Tris-HCl, 361 m
M NaCl, 0.02 mg / ml, polybrene, pH 7.
8 Enzyme concentration: The final concentration of each enzyme is shown in Table 8.

[0128]

[Table 8]

Substrate solution: Enzyme F. G. H. The following substrates f, g, h, and i were used for each of I, and the final concentrations thereof are shown in Table 8. f. H-D-Phe-Pip-Arg-pNA (Kabi V
manufactured by itrum) g. HD-Val-Leu-Lys-pNA (Kabi V
manufactured by itrum) h. HD-Pro-Phe-Arg-pNA (Kabi V
made by itrum)

[0130]

[Chemical 1]

(2) Method The (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI solution obtained in Example 9 was added to the wells of a 96-well microplate for ELISA, and then the enzyme solution was added, and the mixture was heated at 37 ° C. for 10 minutes.
Keep warm for minutes. Next, add the substrate solution and develop color at 37 ° C.
Absorbance was measured at 405 nm.

SLPI was used as a control.

(3) Results (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI and SLPI showed no inhibitory activity against all of the above four enzymes at a final concentration of 1 × 10 ^-5 M or less.

[0134]

Example 12 (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI inhibitor constant (K
i) The value measuring method is shown below.

(1) Reagent Solution Buffer: 0.1M N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES),
1.0 M NaCl 0.1% polyethylene glycol (pH 7.5) Enzyme solution: Each of the following enzymes is dissolved in the above buffer solution to a concentration 9 times that shown in Table 9. (A) Human sputum-derived polymorphonuclear leukocyte elastase (EPC
(Funakoshi Pharmaceutical Co., Ltd.) (b) Bovine spleen trypsin (manufactured by Sigma) Substrate solution: The substrate of the following (d) and (e) for each of the above enzymes (a) and (b) at the concentrations shown in Table 9 In (d), DMSO: the above buffer solution is dissolved 1: 9, and (e) is dissolved in DMSO to make a substrate solution. (D) MeO-Suc-Ala-Ala-Pro-Va
l-pNA (manufactured by Bachem) (e) Bz-Arg-pNA (manufactured by Bachem)

[0136]

[Table 9]

(2) Method 120 μl was added to the well of the 96-well microplate for ELISA.
Buffer, and then 20 μl of sample solution of (Arg ⁵⁹ —Ala ¹⁰⁷ ) SLPI obtained in Example 9 was added, followed by 20 μl of enzyme solution and the mixture was added to 2
The reaction was carried out at 5 ° C for 1 hour. Next, 20 μl of substrate solution was added to develop color at 25 ° C., and the absorbance was measured at 405 nm. SLPI was used as a control.

From this result, the method of Henderson et al. (Hender
son, PJF (1972), Biochem. J. 127 , 321-3
33) according to the present invention, (Arg ⁵⁹ —Ala ¹⁰⁷ ) SL
The PI inhibition constant (Ki) value was determined. The results are shown in Table 10.

From Table 10, the active form (A
The relative activity ratio of the trypsin inhibitory activity to the elastase inhibitory activity of rg ⁵⁹ -Ala ¹⁰⁷ ) SLPI (inhibition constant ki (T) for trypsin / inhibition constant ki (E) for elastase) was about 14,000. That is, (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI did not substantially exhibit trypsin inhibitory activity.

[0140]

[Table 10]

[0141]

[Example 13] SLPI using human sputum-derived polymorphonuclear leukocyte elastase
Treatment of human sputum polymorphonuclear leukocyte elastase (manufactured by EPC,
Funakoshi Chemical Co., Ltd. and SLPI were respectively buffered (0.
1M HEPES, 1.0M NaCl, 0.1% PE
GLP, pH 7.5) dissolved and SLP of constant concentration
An elastase solution was added to the solution I, and the mixture was kept warm at 37 ° C. After each of these samples was collected in a timely manner, Tris-HC
l buffer (pH 6.8), SDS, 2-mercaptoethanol, glycerol, Coomassie Brilliant Blue at final concentration of 50 mM, 4 w / v%, 2 v / v
%, 12v / v%, 0.01w / v% so that T
SDS-polyacrylamide gel electrophoresis using ricine (Sigma) as an electrode buffer (Schagger,
H., Anal. Biochem. 166 , 368, 1987). The gel after electrophoresis is Coomassie Brilliant Blue G
-250, 50v / v% methanol / 10v / v
Decolorized with% acetic acid.

(1) To 100 μM SLPI, elastase was added at a final concentration of 200 μM, and 3 times until 192 hours.
The results of keeping the temperature at 7 ° C are shown in Fig. 9. Samples were taken over time. SLPI (SDS of lane 2 band a in FIG. 9)
-Molecular weight of about 14,000 on PAGE)
It was significantly reduced after 5 minutes. Along with that, band b
(Molecular weight: about 11,000) appeared from 45 minutes and was observed up to 6 hours. In addition, the band c having a molecular weight of about 5,500 gradually increased from the 45th minute, and showed a substantially constant band density from 6 hours to 8 days at 37 ° C.

(2) To 50 μM of SLPI, elastase was diluted 2-fold from the final concentration of 200 μM to 3.13.
FIG. 10 shows the results when the mixture was incubated at 37 ° C. for 1 hour after adding 7 concentrations up to μM. Band e of lane 2 in FIG.
(Molecular weight about 14,000) indicates the band of SLPI. As is clear from FIG. 10, SLPI 50 μM
On the other hand, when elastase was used at a final concentration of 100 μM or more, remarkable degradation of SLPI in band e was observed. The band f (molecular weight of about 11,000) and the band g (molecular weight of about 5,500) were apparently novel bands, and they appeared with the disappearance of the band e, and were considered to be SLPI degradation products.

(3) Next, the polypeptide in the polyacrylamide gel thus obtained was treated with Milli Blot-SDE.
Using a device (Millipore), the membrane (Im
mobilon-P ^SQ , Millipore). After transfer, membrane is 0.1w / v% Coomassie blue / 40v / v
It was dyed with% methanol / 1v / v% acetic acid and washed with water. After air-drying, cut out the band of interest and apply it to Applied Biosystems
Protein sequencer (Applied Biosystems; 4
77A), the amino acid is cleaved from the N-terminus and converted to PTH, and then PTH-amino acid analyzer (Applied Biosyste
The product was analyzed by MS, 120A) and the N-terminal amino acid sequence was determined. The results are shown in Table 11.

[0145]

[Table 11]

From Table 11, bands b and f are amino acid sequences from ALA ¹⁶ of SLPI, and bands c and g are A.
It was completely consistent with the amino acid sequence of from rg ^58. Therefore, it was confirmed that the selective cleavage site of SLPI in human sputum-derived elastase was between Ser ¹⁵ -Ala ¹⁶ and Thr ⁵⁷ -Arg ⁵⁸ .

Based on the N-terminal amino acid sequences of bands b and f, and bands c and g (Table 11), the degradation pattern and the molecular weight (FIGS. 9 and 10), the respective polypeptides are as shown in Table 12 below. It was confirmed to be a polypeptide.

[0148]

[Table 12]

[0149]

Example 14 Normal human saliva and human sputum-derived polymorphonuclear leukocyte elastase
Simultaneous treatment of SLPI with (1) SLPI and human sputum-derived polymorphonuclear leukocyte elastase (manufactured by EPC, Funakoshi Pharmaceutical Co., Ltd.) were ultrafiltered with a 0.22 μm filter (Millex GS, Millipore) in normal human saliva, Each was dissolved in a final concentration of 50 μM. This mixed solution was kept warm at 37 ° C., collected over time, and analyzed by SDS-PAGE as in Example 1.
The result is shown in FIG. In the figure, when SLPI (molecular weight of about 14,000) shown in Lane 2 band I was incubated at 37 ° C in saliva containing the same concentration of elastase,
At 96 hours, a new band J having a molecular weight of about 5,500 appeared.

(2) Next, this band J was electrically transferred to a membrane in the same manner as in Example 1, and then the N-terminal amino acid sequence was analyzed. The results are shown in Table 13. From this result, the N-terminal sequence of band J was completely in agreement with the SLPI amino acid sequence of Arg ^{58 and} later.

From the above, it was confirmed that SLPI is cleaved between Thr ^{57 and} Arg ⁵⁸ in normal human saliva, which is exocrine fluid, and in the presence of elastase at the same concentration as SLPI. From the molecular weight of band J in FIG. 11 (about 5,500) and the N-terminal amino acid sequence in Table 13, it was confirmed that band J is (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI polypeptide.

[0152]

[Table 13]

[0153]

Example 15: Normal human saliva and human sputum-derived polymorphonuclear leukocyte elastase
Simultaneous treatment of (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI by (1) In the same manner as in Example 14, (A)
sn ⁵⁵ -Ala ¹⁰⁷ ) SLPI and human sputum-derived polymorphonuclear leukocyte elastase at final concentrations of 50 μM and 10 respectively.
It was dissolved in 0 μM, and this mixed solution was kept warm at 37 ° C.
This sample was collected over time and analyzed by SDS-PAGE.

The results are shown in FIG. (Asn ^55- A
la ¹⁰⁷ ) SLPI (molecular weight about 5,800, band L)
Is treated with normal human saliva and elastase, the band M having a slightly lower molecular weight than the band L is 3
It newly appeared at 12 ° C at 7 ° C. The band L gradually decreased with the extension of the heat retention time, and after the 48th hour, almost all of the band M remained.

(2) Next, the N-terminal amino acid sequence of this band M was analyzed in the same manner as in Example 1. The results are shown in Table 14.

From this result, the N-terminal amino acid sequence of band M was the SLPI amino acid sequence after Arg ⁵⁸ .

From the above, (Asn ⁵⁵ -Ala ¹⁰⁷ ) SL
PI was cleaved between Thr ^{57 and} Arg ⁵⁸ in the simultaneous treatment of normal human saliva and elastase, and all (Arg
⁵⁸ -Ala ¹⁰⁷ ) It was confirmed that it could be converted to SLPI.

[0158]

[Table 14]

[0159]

Example 16 Human sputum-derived polymorphonuclear leukocyte elastase (Asn
Treatment of ^55- Ala ¹⁰⁷ ) SLPI In the same manner as in Example 13, a final concentration of 100 μM (A
sn ⁵⁵ -Ala ¹⁰⁷ ) SLPI final concentration of 200
It was treated with μM elastase at 37 ° C. for 48 hours.

After separating this sample by SDS-PAGE,
In the same manner as in Example 1, electrically transferred to a filter, cut out a band corresponding to a molecular weight of about 5,500,
The N-terminal amino acid sequence was analyzed. The results are shown in Table 15.

From this result, it was confirmed that the Eras
Treated with Tase (Asn⁵⁵-Ala ¹⁰⁷) SLPI N
The terminal amino acid sequence is Arg⁵⁸Subsequent SLPI amino acids
Converted to an array.

From the above, (Asn ⁵⁵ -Ala ¹⁰⁷ ) SL
PI is Thr ^{57-based on} human sputum-derived elastase.
Cut between Arg ⁵⁸ and all (Arg ^58- Al
it was confirmed to be converted to a ¹⁰⁷⁾ SLPI.

[0163]

[Table 15]

[0164] From the results of Examples 13~16 (Arg ⁵⁸ -
It is found that Ala ¹⁰⁷ ) SLPI is the native SLPI carboxy-terminal polypeptide.

[0165]

Example 17 Human growth hormone fragment polypeptide (Arg ⁵⁸ -Ala
¹⁰⁷ ) Fusion protein expression plasmid of SLPI polypeptide
And preparation of its transformant (Met ^-1 Phe ¹ -Phe ¹³⁹ ) human growth hormone fragment polypeptide and fusion protein expression plasmid pGH-of the SLPI polypeptide having the enterokinase cleavage sequence gene (Arg ⁵⁹ -Ala ¹⁰⁷ ). Polymerase chain reaction: PCR method (Saiki, using E59 as a template DNA, synthetic DNA fragment, (FIG. 13) as a primer)
RK, Gelfand, DH, Stoffel, S., Schare, SJ, Hi
guchi, R., Hoen, GT, Mullis, KB & Erlich, HA;
Science, 239, 487-491, 1985), and the amplified product was resynthesized into a synthetic DNA fragment,
After amplification by PCR using (Fig. 13) as a primer, it was cleaved with BglII (Takara Shuzo) to obtain a fragment of about 120 bp. Note that pGH-E59 was obtained in Example 6.

The PCR conditions were such that after denaturing at 93 ° C. for 5 minutes, one cycle was performed at 93 ° C. for 1.5 minutes, 57 ° C. for 2 minutes, 72 ° C. for 2 minutes, and 30 cycles were carried out.
The reaction was stopped at 7 ° C for 7 minutes.

On the other hand, after cutting pGH-E59 with BglII (Takara Shuzo), about 4.6 kbp BglII-BglII fragment was separated by agarose gel electrophoresis.
It was recovered using LEAN II (manufactured by BIO 101). The BglII-BglII fragment of about 4.6 kbp was dephosphorylated at the 5'end using Bacterial Alkaline Phosphatase (Takara Shuzo) according to the Takara Shuzo protocol.

Dephosphorylated about 4.6 kbp BglII
-BglII fragment and about 115 bp amplified by PCR method
The BglII-BglII fragment of E. coli was subjected to a ligation reaction using a ligation kit (Takara Shuzo), and then E. coli HB1
01 competent cells (Takara Shuzo) and cultured on LB agar medium containing ampicillin to obtain a transformant (HB / GH-T58) containing the desired fusion protein expression plasmid. Extraction of plasmid DNA from the transformant, QIAG
Was purified by EN (manufactured by BIO101), were obtained with the human growth hormone fragment polypeptides and thrombin cleavage sequence gene (Arg ⁵⁸ -Ala ¹⁰⁷⁾ SLPI polypeptide of the fusion protein expression plasmid pGH-T58 (Figure 1
4).

The nucleotide sequence was determined by using Dye Terminetor kit with plasmid DNA as a template and synthetic DNA as a primer.
(Hitachi) was used to directly confirm with a fluorescent automatic DNA sequencer SQ3000 manufactured by Hitachi.

[0170]

EXAMPLE 18 Fusion obtained in Expression Example 17 of the fusion protein gene protein gene expression plasmid p
E. coli HB101 strain containing GH-T58 was added to 0.2
5% glucose, 3 mg / ml yeast extract (DIFCO
Modified M9 medium (1% (NH ₄ ) ₂ HPO ₄ -0.3) containing 20 mg / ml casamino acid (manufactured by DIFCO).
% K ₂ SO ₄ -0.3% NaCl) was autoclaved and then inoculated into 500 ml of a culture solution in which MgSO ₄ and CaCl ₂ were aseptically added so that the final concentration was 1 mM, and the cells were cultured overnight.

This overnight culture solution was used as the same culture solution 1 as above.
To 0 liter, OD660 was added to be 0.2, and jar fermenter culture was performed at 37 ° C. O
When D660 reached 20, 3-β-indoleacrylic acid was added to the culture medium so that the final concentration was 150 μg / ml, and the culture was further continued at 37 ° C. for 4 hours. After that, E. coli cells were collected by centrifugation and washed with 0.5 M Tris buffer (pH 8.0).

The washed cells were suspended in 0.5 M Tris buffer containing lysozyme (1/200 of wet weight of cells) and EDTA.3Na (1/20 of wet weight of cells) and sonicated. Generator (BRANSON, CELL DISRU
After disrupting the cells using PTOR 900), centrifuge to separate the desired fusion protein into the precipitate (inclusion body).
obtained as y).

E. coli H containing pGH-T58
B101 strain (HB / GH-T58)
No. 62 (FERM BP-3862) has been deposited internationally on May 20, 1992 at the Institute for Microbial Industry, Institute of Industrial Technology.

[0174]

Example 19 Sulfonation of Cysteine Residue of Fusion Protein and Sulfonated Fusion Protein
Cleavage reaction with white thrombin After suspending 25 g of the fusion protein obtained in Example 18 in 200 ml of 0.5 M Tris buffer (pH 8.0),
240 g of urea was added for solubilization, and then the final concentration was 0.7.
Sodium sulfite (Wako Pure Chemical Industries, Ltd.) was added so as to give M and reacted at 45 ° C. for 20 minutes. Furthermore, sodium 4-thionate (Sigma) was added to a final concentration of 0.1 M, and the reaction was carried out at 45 ° C. for 20 minutes. The operation of adding 10 mM Tris buffer (pH 8.0) to make the reaction solution 1 liter and then performing ultrafiltration (Fuji filter; FILTRON) until the volume reached 200 ml was repeated 5 times. In this way, the sulfonated fusion protein solution (portion that did not pass through the ultrafiltration membrane) was 300
Add 0 U of bovine thrombin (Mochida Pharmaceutical Co., Ltd.) and add 3 at 37 ° C.
Reacted for hours. Then, it is contained in the solution that has passed through the 300K filter by ultrafiltration (Arg ⁵⁸ -Ala).
¹⁰⁷ ) SLPI sulfonated derivative was analyzed by reverse phase HPLC (VY
Purified by DAC; Plotein C4) and lyophilized. The N-terminal amino acid sequence of the resulting (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI sulfonated derivative was determined by a protein sequencer (Applied Biosystems 477A).

[0175]

Example 20 Activity of (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI Sulfated Derivative
Refolding (disulfide reaction) into a functional molecule (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLP obtained in Example 19
1 g of the I-sulfonated derivative was added to 5 liters of Refolding buffer (0.05 M Tris-HCl, pH 8.5, 0.3).
mM-oxidized glutathione, 1.5 mM reduced glutathione), and reacted at 4 ° C. for 2 days. Then, the reaction solution was subjected to S-Sepharose (Pharmacia) ion exchange chromatography, and its elution pattern is shown in FIG. Show.
The desired fraction, which was eluted with 0.3 M NaCl, was subjected to reverse phase HPL.
After further purification with C (VYDAC, Protein C4),
Lyophilized and activated (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI
Got

[0176]

Example 21 (Asn ⁵⁸ -Ala ¹⁰⁷ ) SLPI serine protector
Measurement of ze inhibitory activity (1) The measuring method is shown below.

(1) Reagent solution Buffer solution: 0.1 M- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES) 0.5
M NaCl, pH 7.5 Enzyme concentration: Each of the following enzymes is dissolved in the above buffer solution to a concentration 9 times that shown in Table 16. A. Human sputum-derived polymorphonuclear leukocyte elastase (manufactured by EPC, Nafukoyaku) B. Bovine spleen trypsin (manufactured by Sigma) C.I. Bovine spleen chymotrypsin (manufactured by Sigma) D. Pig spleen elastase (manufactured by Sigma) E. Human-derived cathepsin G (manufactured by EPC, Funakoshi Chemical Co., Ltd.) Substrate solution: For each of the above enzymes A, B, C, D, and E, the following a, b, c, d, and e substrates have the concentrations shown in Table 16, respectively. DMSO: buffer solution (1:
9) Dissolve in solution (DMSO final concentration 10 in buffer)
%) Substrate solution. a. MeO-Suc-Ala-Ala-Pro-Val
-PNA (manufactured by Bachem) b. Bz-Arg-pNA (manufactured by Bachem) c. Suc-Ala-Ala-Pro-Phe-pNA
(Manufactured by Bachem) d. Suc-Ala-Ala-Ala-pNA (Hanai Chemical) e. Suc-Phe-Pro-Phe-pNA (Bac
made by hem)

[0178]

[Table 16]

(2) Method 120 μl was added to the well of the 96-well microplate for ELISA.
Buffer, then 20 μl of sample solution of (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI obtained in Example 20 was added, followed by 20 μl of enzyme solution and the mixture was added to 3
The reaction was carried out at 7 ° C for 10 minutes. Next, 20 μl of the substrate solution was added, color was developed at 37 ° C., and the absorbance was measured at 405 nm.

SLPI was used as a control, the concentration of the solution was changed, the inhibitory activity was measured, and the inhibitory protein concentration (IC ₅₀ value) showing a 50% inhibitory concentration was determined. The results are shown in Table 17.

[0181]

[Table 17]

[0182]

Example 22 (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI Serine Proteare
(2) In the four types of enzymes shown below, (Arg ⁵⁸ -Ala
¹⁰⁷ ) The inhibitory protein concentration (IC ₅₀ ) value showing 50% inhibition of SLPI and SLPI as a control was determined. F. Human plasma-derived thrombin (manufactured by Sigma) G. Human plasma-derived plasmin (manufactured by Sigma) Human plasma-derived kallikrein (Funakoshi Pharmaceutical, manufactured by Protogen) I. The pig-derived FXa (Daiichi Pure Chemicals) measurement method is shown below. (1) Reagent solution Buffer solution: The buffer solution for each enzyme is shown below.

Thrombin, FXa; 50 mM Tris
-HCl, 7.5 mM EDTA-2Na, 175 mM
NaCl, pH 8.4 plasmin; 50 mM Tris-HCl, 100 mM
NaCl, pH 7.4 kallikrein; 50 mM Tris-HCl, 361 m
M NaCl, 0.02 mg / ml, polybrene, pH 7.
8 Enzyme concentration; The final concentration of each enzyme is shown in Table 18. Substrate solution: Substrates of the following f, g, h, and i were used for each of the above enzymes F, G, H, and I, and their final concentrations are shown in Table 18. f. H-D-Phe-Pip-Arg-pNA (Kabi V
manufactured by itrum) g. HD-Val-Leu-Lys-pNA (Kabi V
manufactured by itrum) h. HD-Pro-Phe-Arg-pNA (Kabi V
made by itrum)

[0184]

[Chemical 2]

[0185]

[Table 18]

(2) Method Example 20 was applied to the holes of a 96-well microplate for ELISA.
The sample solution of (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI obtained in Example 1 was added, and then the enzyme solution was added, and the mixture was mixed with 3
It was kept at 7 ° C for 10 minutes. Next, add the substrate solution to 37 ℃
The color was developed with and the absorbance was measured at 405 nm.

SLPI was used as a control.

(3) Results (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI and SLPI showed no inhibitory activity against all of the above four enzymes at a final concentration of 1 × 10 ^-5 M or less.

[0189]

Example 23 (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI inhibitor constant (K
i) The value measuring method is shown below.

(1) Reagent solution Buffer solution: 0.1M N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES),
1.0 M NaCl 0.1% polyethylene glycol (pH 7.5) Enzyme solution: Each of the following enzymes is dissolved in the above buffer solution to a concentration 9 times that shown in Table 19. (A) Human sputum-derived polymorphonuclear leukocyte elastase (EPC
(Funakoshi Pharmaceutical Co., Ltd.) (b) Bovine spleen trypsin (manufactured by Sigma) Substrate solution: For each of the above enzymes (a) and (b), the substrates of the following (d) and (e) at the concentrations shown in Table 19 In (d), DMSO: the above buffer solution is dissolved at 1: 9, and (e) is dissolved in DMSO so that a substrate solution is obtained. (D) MeO-Suc-Ala-Ala-Pro-Va
l-pNA (manufactured by Bachem) (e) Bz-Arg-pNA (manufactured by Bachem)

[0191]

[Table 19]

(2) Method 120 μl was added to the well of the 96-well microplate for ELISA.
Buffer, then 20 μl of sample solution of (Arg ⁵⁸ —Ala ¹⁰⁷ ) SLPI obtained in Example 20 was added, followed by 20 μl of enzyme solution and the mixture was added to 2
The reaction was carried out at 5 ° C for 1 hour. Next, 20 μl of substrate solution was added to develop color at 25 ° C., and the absorbance was measured at 405 nm. SLPI was used as a control.

From this result, the method of Henderson et al. (Hender
son, PJF (1972), Biochem. J. 127 , 32.
(Arg ⁵⁸ -Al of the present invention according to 1-333).
a ¹⁰⁷ ) The inhibition constant (Ki) value of SLPI was determined. The results are shown in Table 20.

From Table 20, the relative activity ratio of the trypsin inhibitory activity to the elastase inhibitory activity of the active form (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI obtained in Example 20 (inhibition constant ki for trypsin ki (T) / inhibition constant ki for elastase ki) (E)) was about 12,000. That is, (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI showed substantially no trypsin inhibitory activity.

[0195]

[Table 20]

[0196]

[Sequence list]

SEQ ID NO: 1 Sequence length: 50 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Arg Lys Pro Gly Lys Cys Pro Val Thr Tyr Gly Gln Cys Leu Met 1 5 10 15 Leu Asn Pro Pro Asn Phe Cys Glu Met Asp Gly Gln Cys Lys Arg Asp 20 25 30 Leu Lys Cys Cys Met Gly Met Cys Gly Lys Ser Cys Val Ser Pro Val 35 40 45 Lys Ala 50

SEQ ID NO: 2 Sequence length: 49 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Lys Pro Gly Lys Cys Pro Val Thr Tyr Gly Gln Cys Leu Met Leu 1 5 10 15 Asn Pro Pro Asn Phe Cys Glu Met Asp Gly Gln Cys Lys Arg Asp Leu 20 25 30 Lys Cys Cys Met Gly Met Cys Gly Lys Ser Cys Val Ser Pro Val Lys 35 40 45 Ala 49

[Brief description of drawings]

FIG. 1 shows SDS-PAGE of a limited degradation product obtained by treating SLPI with normal human saliva in Example 1. In the figure, the band of the line indicated by a is SLPI (MW about 1
3,000) band, similarly, b is a limited degradation product (Tyr ²¹ -Ala ¹⁰⁷ ) SLPI, and c is a limited degradation product ((Tyr ²¹ -Leu ⁷² ) or (Tyr ²¹ -Met ⁷³ ).
SLPI), d is a polypeptide of the present invention (MW about 5,5
00), e is ((Met ⁷³ -Ala ¹⁰⁷ )) or (Leu
^74- Ala ¹⁰⁷ ) SLPI) band is shown.

FIG. 2 shows (Asn ⁵⁵ —Ala ¹⁰⁷ ) S in Example 2.
SD of limited degradation products when LPI is treated with normal human saliva
Indicates S-PAGE. In the figure, the band of the line indicated by f is (Asn ⁵⁵ -Ala ¹⁰⁵ ) SLPI (MW about 5,8).
00) band, and similarly g indicates the band of the polypeptide of the present invention (MW of about 5,500).

FIG. 3 shows an elution pattern by reverse phase chromatography of the produced polypeptide of the present invention, (Arg ⁵⁹ —Ala ^{10 7} ) SLPI, in Example 3.

FIG. 4 shows the DNA fragment (,) containing the thrombin cleavage sequence gene used in Example 6 (1).

FIG. 5 shows the construction of the fusion protein expression plasmid pGH-T59 in Example 6 (1).

FIG. 6 represents the synthetic DNA fragment (,,) used in Example 6 (2).

FIG. 7 represents the construction of pGH-E59 in Example 6 (2).

FIG. 8 shows an activated form (Arg ⁵⁹ —Ala) in Example 9.
¹⁰⁷ ) shows the elution pattern of SLPI by S-Sepharose ion exchange chromatography.

FIG. 9: SDS-PA obtained by treating SLPI with double the amount of human polymorphonuclear leukocyte elastase in Example 13.
Shows GE. In the figure, the band indicated by a is SL.
A band of PI (MW about 14,000) is shown, b is a limited degradation product (Ala ¹⁶ -Ala ¹⁰⁷ ) SLPI, and c is a band of the polypeptide of the present invention (MW about 5,500). In addition, a group of bands d represents elastase.

FIG. 10 shows SDS-PAGE of a limited degradation product obtained by treating SLPI with varying concentrations of human polymorphonuclear leukocyte elastase in Example 13. Lanes 3 to 9
Is equivalent to the addition of elastase from 3.13 μM to 200 μM to 50 μM SLPI,
5 μl of each lane was electrophoresed. In the figure, the band of the line indicated by e indicates the band of SLPI (MW about 14,000), and f indicates the limited decomposition product (Ala ¹⁶ -Al.
a ¹⁰⁷ ) SLPI, as well as g, indicates the band of the polypeptide of the present invention (MW about 5,500). In addition, a group of bands H represents elastase.

FIG. 11 shows SDS-PAGE of limited degradation products obtained by simultaneously treating SLPI with normal human saliva and human sputum-derived polymorphonuclear leukocyte elastase in Example 14. In the figure, the band of the line indicated by I is SLPI (MW about 1
4,000), and J indicates the band of the polypeptide of the present invention (MW about 5,500). Also,
Band K of the group represents elastase.

FIG. 12 shows (Asn ⁵⁵ —Ala) in Example 15.
¹⁰⁷ ) S of limited degradation products obtained by simultaneously treating SLPI with normal human saliva and human sputum-derived polymorphonuclear leukocyte elastase
Indicates DS-PAGE. In the figure, the band line indicated by ^{L (Asn 55 -Ala 10 7)} SLPI (MW about 5,
800), and M also shows the band of the polypeptide of the present invention (MW about 5,500). Also, a group of bands N represents elastase.

13 shows a synthetic DNA fragment in Example 17 ,.

FIG. 14 shows (Arg ⁵⁸ —Al in Example 17)
a ¹⁰⁷ ) Construction of a fusion protein expression plasmid pGH-T58 of SLPI polypeptide.

FIG. 15 shows an activated form (Arg ⁵⁸ —Al in Example 20).
a ¹⁰⁷ ) shows an elution pattern of SLPI by S-Sepharose ion exchange chromatography.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI Technical display area C07K 15/12 8517-4H C12N 1/21 7236-4B 9/99 15/15 15/62 C12P 21/02 C 8214-4B 21/06 8214-4B // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) C07K 99:00 (72) Inventor Tetsuya Suga 4-3 Asahigaoka, Hino-shi, Tokyo No. 2 Inside the Tokyo Research Center, Teijin Ltd. (72) Akiko Takeuchi 4-chome, Asahigaoka, Hino City, Tokyo No. 2 Inside the Tokyo Research Center, Teijin Ltd. (72) Yoichi Matsumoto 4-chome, Asahigaoka, Hino City, Tokyo No. 2 inside Tokyo Research Center, Teijin Limited

Claims (28)

[Claims] 1. An elastase-inhibiting polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or a part thereof having elastase-inhibiting activity, or an amino acid sequence biologically equivalent to them, or a homopolymer thereof. 2. The elastase-inhibiting activity polypeptide or a homopolymer thereof according to claim 1, wherein Cys residues in the amino acid sequence mutually form a disulfide bond necessary for inhibitory activity. 3. The disulfide bond has Cys ⁶⁴ and Cys ⁹³ , Cys ⁷¹ and Cys ⁹⁷ , Cys in the amino acid sequence.
The elastase inhibitory activity polypeptide or a homopolymer thereof according to claim 2, which is a bond between ⁹² and Cys ⁸⁰ , and Cys ⁸⁶ and Cys ¹⁰¹ with each other. 4. The elastase inhibitory activity polypeptide or homopolymer thereof according to any one of claims 1 to 3, which is obtained by a gene recombination technique. 5. An elastase-inhibiting polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or a part thereof having elastase-inhibiting activity, or an amino acid sequence biologically equivalent thereto, or a homopolymer thereof. 6. The elastase-inhibiting activity polypeptide or a homopolymer thereof according to claim 5, wherein Cys residues in the amino acid sequence mutually form a disulfide bond necessary for inhibitory activity. 7. The disulfide bond has Cys ⁶⁴ and Cys ⁹³ , Cys ⁷¹ and Cys ⁹⁷ , Cys in the amino acid sequence.
7. The elastase-inhibiting active polypeptide or a homopolymer thereof according to claim 6, which is a bond between Cys residues of ⁹² and Cys ⁸⁰ , and Cys ⁸⁶ and Cys ¹⁰¹ . 8. The elastase-inhibiting polypeptide or a homopolymer thereof according to any one of claims 5 to 7, which is obtained by a gene recombination technique. 9. The following formula [I] AX—B ... [I] (wherein A represents a carrier protein; B represents the elastase-inhibiting polypeptide or its homologue according to claim 1 or 5). X represents a cross-linked polypeptide having an amino acid sequence that can be cleaved by a chemical method or a biological method under conditions that do not denature the target protein). 10. The fusion protein according to claim 9, wherein the carrier protein is human growth hormone or a polypeptide fragment thereof. 11. The carrier protein is Phe from amino acid residue number 1 position to Phe position 139 of natural human growth hormone.
10. The fusion protein according to claim 9, which is a polypeptide fragment up to. 12. The carrier protein comprises Phe from the amino acid residue number 1 position to Gln from the 122 position amino acid of natural human growth hormone.
10. The fusion protein according to claim 9, which is a polypeptide fragment up to. 13. The carrier protein is T7 phage gene1.
10. The fusion protein according to claim 9, which is 0 protein or a polypeptide fragment thereof. 14. X is (Asn-Gly) _n , (Val
-Pro-Arg) _n , (Leu-Val-Pro-A
rg _nn ) _n , and (Asp-Asp-Asp-Asp-
Lys) _n (n represents an integer of 1 to 10), which is a bridging peptide or polypeptide having an amino acid sequence selected from the group consisting of Gly, Arg, and Arg of X, respectively. The fusion protein according to claim 9, which is bound to either Lys or Lys. 15. A method for producing the elastase-inhibiting activity polypeptide or a homopolymer thereof according to claim 1 or 5, wherein the fusion protein represented by the above formula [I] is treated by a chemical method or a biological method. A method consisting of processing by. 16. The method of claim 15, wherein the chemical method is with hydroxylamine or a homologue thereof. 17. The method according to claim 15, wherein the biological method is thrombin, enterokinase or a homologue thereof. 18. A gene encoding a carrier protein and an elastase inhibitory activity having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof having an elastase inhibitory activity or an amino acid sequence biologically equivalent to them. The gene that encodes the polypeptide is
A fusion protein gene linked via a gene encoding a cross-linking peptide having an amino acid sequence cleavable by a chemical method or a biological method under the condition that the target protein is not denatured or a homopolymer thereof. 19. An amino acid sequence cleavable by a chemical method or a biological method under the condition that the target protein is not denatured is (Asn-Gly) _n , (Val-Pro-Ar).
g) _n , a bridge having an amino acid sequence selected from the group consisting of (Leu-Val-Pro-Arg) _n , and (Asp-Asp-Asp-Asp-Lys) _n (n represents an integer of 1 to 10) The fusion protein gene according to claim 18, which is a peptide or a polypeptide. 20. The carrier protein is human growth hormone, T7.
The fusion protein gene according to claim 18, which is a phage gene10 protein or a polypeptide fragment thereof. 21. The carrier protein is Phe at amino acid residue number 1 to Phe at amino acid residue number 1 of natural human growth hormone.
The fusion protein gene according to claim 18, which is a polypeptide fragment up to. 22. A fusion protein gene expression-type plasmid containing the fusion protein gene according to any one of claims 18 to 21. 23. A recombinant microbial cell containing the fusion protein gene according to any one of claims 18 to 21. 24. A method for producing the elastase inhibitory activity polypeptide or a homopolymer thereof according to claim 1 or 5, wherein human leukocyte elastase inhibitory protein (SLPI) is enzymatically treated, or SEQ ID NO: 1 or sequence. A method comprising chemically synthesizing using an amino acid constituting the amino acid sequence represented by No. 2. 25. A pharmaceutical preparation comprising a therapeutically effective amount of the elastase inhibitory active polypeptide or its homopolymer according to claim 1 or 5, and a pharmaceutically acceptable carrier. 26. A disease caused by neutrophil hyperactivation, comprising a therapeutically effective amount of the elastase-inhibiting activity polypeptide or homopolymer thereof according to claim 1 or 5, and a pharmaceutically acceptable carrier. Therapeutic agent. 27. The therapeutic agent according to claim 26, wherein the disease is caused by neutrophil-releasing protease. 28. The therapeutic agent according to claim 27, wherein the disease is an inflammatory disease, platelet aggregation thrombosis, or post-ischemic reperfusion injury.