JP2798573B2 - Natural polypeptide having human neutrophil elastase inhibitory activity and pharmaceutical preparation containing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a natural polypeptide having human neutrophil elastase inhibitory activity and a pharmaceutical preparation containing the same. More particularly, elastase inhibitory activity polypeptide de having the amino acid sequence or their biologically equivalent amino acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, a process for their preparation, and pharmaceutical and their therapeutic effective amount A pharmaceutical formulation comprising a pharmaceutically acceptable carrier.

[0002]

BACKGROUND OF THE INVENTION Chronic bronchitis, diffuse panbronchiolitis, bronchiectasis, pulmonary emphysema, ARDS, bacterial pneumonia, bronchial asthma, etc. are diseases in which neutrophils increase in sputum or bronchial and alveolar lavage fluid. Known (Am. Rev. Respir.
Dis., 132 , 417, 1985; Am. Rev. Respir. D
, 142 , 57, 1990), neutrophils migrating into the lungs and bronchus release elastase (Am. Rev. Respir. Dis., 141 , 689, 19).
90) Therefore, it has been reported that high concentrations of neutrophil-derived elastase are detected in airway 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).

[0003] Neutrophil proteases are originally foreign foreign substances,
Although it has a role as a biological defense mechanism against bacteria, viruses, etc., if neutrophil protease is present in excess, it attacks itself and the lung matrix is degraded (J. Biol. Chem., 255) . J., 8848, 1980;
Biol. Chem., 255 , 12006, 1980).

[0004] Protease inhibitors are prepared by the living body against this excess protease, but the balance between neutrophil protease and protease inhibitor is lost, and when neutrophil protease becomes excessive, the lung matrix becomes large. It is believed that the illness will be severe if the patient is injured and this 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).

[0005] 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 in airway secretory gland cells, but is present in excess in localized inflammation The mode of degradation of SLPI by neutrophil elastase has not been elucidated.

[0006] SLPI is also present in human saliva, nasal discharge, semen, uterine mucus, etc., and is isolated from parotid gland secretions.
The amino acid sequence of PI was analyzed (Proc. Natl. Aca
.. d Sci USA, 83, 6692,1986; JP-T Akira 62
This protein gene has been isolated from a human parotid gland gene library and its structure has been determined (Nucl. Acid. Res. 14 , 7883, 198).
6; JP-T-62-501262). However, SLP present in human saliva, nasal discharge, semen, uterine mucus, etc.
The mode of degradation of I has not been elucidated.

[0007] SLPI not only inhibits chymotrypsin-like enzymes such as human neutrophil elastase, human cathepsin G, human pancreatic elastase and human chymotrypsin, but also inhibits physiologically important trypsin-like enzymes such as trypsin. have. Due to this trypsin-like enzyme inhibitory activity, SLPI has been difficult to use as a therapeutic agent 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 of the SLPI by genetic engineering techniques has the S
It has already been confirmed that LPI has substantially no trypsin inhibitory activity while maintaining elastase inhibitory activity (Biotech, 7 , 55, 1989; Protein Engine
ering, 3 , 215, 1990; WO 89/06239).

However, although these SLPI carboxy-terminal polypeptides have been successful in removing trypsin-like enzyme inhibitory activities that are expected to lead to side effects, they are not suitable for use in pharmaceutical preparations for therapeutic purposes. This can 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, they produce antigenicity and produce antibodies. Is a concern.

[0009] When an antibody against a polypeptide preparation is generated, the antibody inhibits the effect of the preparation, not only losing the drug effect, but also causing a rapid antigen-antibody reaction in the living body. Administration of the formulation to the patient will not be possible after antibody production.

[0010] On the other hand, it is known that humans are particularly liable to produce antibodies against non-natural polypeptides, and the use of a non-natural SLPI carboxy-terminal polypeptide which has been reported so far is highly dangerous. Should be avoided.

On the other hand, a natural polypeptide already existing in a living body is a so-called self-polypeptide,
Antibody production is not a concern and is clearly superior as a formulation for human administration.

[0012]

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

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

Although the mode of degradation of SLPI present in saliva has not been elucidated, fragments degraded by enzymes in saliva are natural polypeptides existing in the living body, that is, self-polypeptides. Don't worry.

The present inventors have made intensive efforts to provide such an elastase inhibitory protein, and as a result, natural SLPI produced by genetic engineering is subjected to specific limited degradation by the enzyme of neutrophil elastase. thing,
The polypeptide subjected to this limited degradation, that is, the amino acid sequence represented by SEQ ID NO: 1 or a polypeptide having a biologically equivalent amino acid sequence has excellent elastase inhibitory activity, cathepsin G inhibitory activity, and trypsin inhibitory activity. The inventors have found that they do not substantially show the activity, and have reached the present invention.

Also, the fact that natural SLPI produced by genetic engineering is subjected to specific limited degradation by enzymes in normal human saliva, and the polypeptide subjected to this limited degradation, that is, the amino acid sequence represented by SEQ ID NO: 2 Or that the polypeptide having a biologically equivalent amino acid sequence has excellent elastase inhibitory activity, cathepsin G inhibitory activity, and does not substantially exhibit trypsin inhibitory activity, and has reached the present invention. It is.

Conventionally, carboxymethylated linear SL
It has been reported that PI was limitedly degraded with an enzyme (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.
Naturally, the resulting fragment is also a non-natural SLPI, and neither describes nor suggests elastase inhibitory activity, cathepsin G inhibitory activity, trypsin inhibitory activity or antigenicity.

[0018]

That is, the present invention relates to 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 a biologically equivalent amino acid sequence. de, the following formula [I] A-X-B ... [I] ( in the formula, A human growth hormone or a portion thereof, is
Or from T7 phage gene10 protein or a part thereof
Represents composed carrier protein; B is elastase inhibitory activity poly <br/> having a portion thereof or a biologically equivalent amino acid sequence having the amino acid sequence or elastase inhibitory activity represented by SEQ ID NO: 1 or SEQ ID NO: 2 represent peptides or Ranaru desired protein; X is under conditions that do not denature the said purpose protein, represents a cross-linked polypeptide having an amino acid sequence cleavable by chemical methods or biological methods. ) Represented by fusion proteins, methods of producing such elastase inhibitory activity polypeptide de, a gene encoding a carrier protein, a portion thereof or a biologically having the amino acid sequence or elastase inhibitory activity represented by SEQ ID NO: 1 or SEQ ID NO: 2 to genes genes encoding elastase inhibitory activity polypeptide having an equivalent amino acid sequence, which encodes the crosslinked peptide having an amino acid sequence cleavable by chemical methods or biological methods under conditions which do not denature the desired protein fusion protein gene linked via a such fusion protein gene fused protein gene expression plasmid or recombinant microbial cells containing, and such elastase inhibitory activity polypeptide de Osamu <br/> care effective amount and a pharmaceutically tolerated in that ing from a carrier, due to neutrophil overactivation sputum Of these, for example, such as elastase and / or cathepsin G, it is a therapeutic agent for a disease caused by neutrophils release proteases.

Accordingly, the present invention relates to an elastase-inhibiting 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 a biologically equivalent amino acid sequence. to provide a de.

In the present invention, the term "biologically equivalent amino acid sequence" refers to an elastase inhibitory activity and a cathepsin G inhibitory activity which are the object of the present invention, exhibit substantially no trypsin inhibitory activity, and have an antigen for humans. In the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, having the property of being non-active or substantially reduced,
The addition or deletion of one or more amino acids and / or
Or substitutions were made elastase inhibitory activity polypeptide de
The say.

Here, "substantially does not show trypsin inhibitory activity" means that trypsin inhibitory activity, which is an object of the present invention, is hindered when used as a therapeutic agent for human diseases due to elastase inhibitory activity and cathepsin G inhibitory activity. It means that almost no physiological action caused by the activity occurs. For example, when compared with the trypsin inhibitory activity of natural SLPI in the measurement method of the example of the present invention, its trypsin inhibitory activity is about 1/10 or less. In addition, the trypsin inhibitory activity of the protein of the present invention is about 1/100 of its elastase inhibitory activity.
100 or less.

It should be noted that a plurality of the elastase inhibitory polypeptides of the present invention have Cys in the amino acid sequence thereof.
Orientation of dimers etc. through disulfide bonds via residues
May produce sesame or the like .

[0023] Such elastase inhibitory activity poly <br/> peptide of the present invention, a portion thereof having an amino acid sequence or elastase inhibitory activity represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a biologically equivalent amino acid sequence Having. As is apparent from the amino acid sequence represented by SEQ ID NO: 1, the polypeptide of the present invention is a Cys residue in which Cys residue is not subjected to carboxymethylation or the like. C
Those in which the ys residues mutually form a disulfide bond necessary for the inhibitory activity of the present invention. Above all, 4
Those having two disulfide bonds are preferred.

Hereinafter, when the elastase-inhibiting polypeptide of the present invention is expressed, the elastase-inhibiting polypeptide represented by SEQ ID NO: 1 is represented by (Arg ⁵⁸ -Al
a ¹⁰⁷ ) may be expressed as SLPI.
The amino acid sequence of ( ^58- Ala ¹⁰⁷ ) corresponds to (Arg ¹ -Ala ⁵⁰ ) in the sequence listing of SEQ ID NO: 1. Therefore, as those having four disulfide bonds, for example, like the disulfide bond in natural SLPI,
^{Cys 64 -Cys 93, Cys 92 -Cys} 80, Cys 71 -
Cys ⁹⁷ and Cys ⁸⁶ -Cys ¹⁰¹ , that is, Cys ⁷ -Cys ³⁶ and Cys ³⁵ in the sequence listing of SEQ ID NO: 1.
-Cys ²³ , Cys ¹⁴ -Cys ⁴⁰ , and Cys ²⁹ -Cy
corresponding to s ^44, it may be mentioned as being polypeptide preferably has four disulfide bonds.
However, as long as having inhibitory activity and reduced antigenicity is an object of the present invention may be a polypeptide de having a disulfide bond or at least 1 pc.

The elastase-inhibiting polypeptide represented by SEQ ID NO: 2 is represented by (Arg ⁵⁹ -Ala ¹⁰⁷ ) SL using the N-terminal number of the amino acid sequence of natural SLPI.
PI (Arg ⁵⁹ -Ala)
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 as in the case of disulfide bonds in natural SLPI
^{-Cys 93, Cys 92 -Cys 80,} Cys 71 -Cy
s ^97, and Cys ⁸⁶ -Cys ^101, i.e., in the sequence listing SEQ ID NO: ^{2, Cys 6 -Cys 35, Cys} 34 -C
ys ^22, Cys ¹³ -Cys ^39, and Cys ²⁸ -Cys ⁴³
Polypeptides having four disulfide bonds, corresponding to the above, can be mentioned as preferred. However, as long as having inhibitory activity and reduced antigenicity is an object of the present invention may be a polypeptide de having a disulfide bond or at least 1 pc.

The present invention further provides the following formula [I] AXB... [I] useful as an intermediate for the production of the polypeptide, wherein A is human growth hormone or a part thereof.
Or from T7 phage gene10 protein or a part thereof
Represents composed carrier protein; B is a portion thereof or a biologically elastase inhibitory activity polypeptide or elastase inhibitory activity having the same amino acid sequence having the amino acid sequence or elastase inhibitory activity represented by SEQ ID NO: 1 or SEQ ID NO: 2 As a part worth having
Or represents Ranaru desired protein; X is under conditions that do not denature the said purpose protein, represents a cross-linked polypeptide having an amino acid sequence cleavable by chemical methods or biological methods. ) Is provided.

[0027] The present invention also provides a method which comprises a process for the preparation of elastase inhibitory activity polypeptide de above, a fusion protein represented by the formula [I], treated by chemical or biological methods, and a method for producing elastase inhibitory activity polypeptide de above, provides a method which comprises chemically synthesized using amino acids comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

The present invention further relates to human growth hormone or
A part of it, or the T7 phage gene10 protein
Or a gene encoding a carrier protein consisting of
A gene encoding 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 elastase inhibitory activity polypeptide having an amino acid sequence biologically equivalent thereto,
Crosslinked peptides having an amino acid sequence that can be cleaved by chemical or biological methods under conditions that do not denature the target protein
And a fusion protein gene-containing plasmid or a recombinant microbial cell containing the fusion protein gene linked through a gene encoding the fusion protein gene.

The present invention further provides a pharmaceutical preparation comprising a therapeutically effective amount of the above elastase inhibitory polypeptide or a homopolymer thereof and a pharmaceutically acceptable carrier, and a disease caused by neutrophil hyperactivation, In particular, the present invention provides 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 by means of gene recombination known in the art. A plasmid expressing the fusion protein is constructed using the fusion protein gene, and the plasmid is introduced into a microbial host cell, for example, Escherichia coli, followed by culturing the microbial cell. It can be obtained by a method similar to the method of 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, the carrier protein represented by A and X
Examples of the cross-linked polypeptide represented by
Examples exemplified in 9/06239 can be given. As the carrier protein represented by A, human growth hormone and a part thereof or T7 phage gene 10 protein (Stu) which have a high expression level of the fusion protein and are easily isolated and purified because they are present as inclusion bodies in microbial cells.
dier, F .; et al, J.A. Mol. Bio
l. 189, 113, 1986) and parts thereof
Can be Human growth hormone also includes methionyl human growth hormone and variants whose amino acid sequence is partially different from the natural one. When a part of human growth hormone is used as a carrier protein, the polypeptide fragment from Phe at amino acid residue 1 to Phe at position 139 and the polypeptide fragment from Phe at position 1 to Gln at position 122 of human growth hormone are used. preferable.

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

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

[0034] Such is the elastase inhibitory activity polypeptide de or Ranaru desired protein represented by crosslinked polypeptide B represented by each of X, Arg and X at the amino terminus of the said purpose protein Gly, Arg, Arg, or Lys Either of them is combined.

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

When such cleavage is carried out by enzymatic treatment, a method using an endopeptidase which 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 a homolog thereof can be used. Examples of such a thrombin or homolog thereof include human thrombin, bovine thrombin, equine thrombin, and pig thrombin. In this case, X is Val-Pro-
It is preferable to use Arg, Leu-Val-Pro-Arg, or a repetitive sequence thereof. Enterokinase or a homolog thereof may also be used. Examples of such enterokinase or homolog thereof include bovine enterokinase for releasing enkephalin from β-galactosidase (VN Dobrynin et al., Bioory-Khim
13 , 119-121, 1987), enterokinase for releasing human atrial natriuretic factor from chloramphenicol acetyltransferase (A. Hobden et al., WPI 87-088509 / 13), and the like. In this case, X is, for example, A
It is preferable to use sp-Asp-Asp-Asp-Lys, or a repetitive sequence thereof.

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

The fusion protein of the present invention represented by the formula [I] is produced by a known means of genetic 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 subjected to chemical methods or 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 of constructing 9) is shown in FIG.

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

A plasmid (pGH-) expressing a fusion protein in which a (Met ^-1 Phe ¹ -Phe ¹³⁹ ) human growth hormone fragment and an (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI fragment polypeptide are linked via a thrombin cleavage sequence. T5
FIG. 14 shows a method for constructing 8).

The (Met ^-1 Phe ^-1 -Phe ¹³⁹ ) human growth hormone fragment and the (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI fragment polypeptide were (Asp-Asp-Asp-Asp)
-Lys) 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 the 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 amplified product was cut with BglII to obtain a human growth hormone fragment. -Cross-linked polypeptide-(Arg ⁵⁸
-Ala ¹⁰⁷⁾ to 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 a Trp promoter, and the 5′-terminal dephosphorylation reaction of this fragment is performed. The thus obtained two fragments are ligated to obtain a fusion protein expression type plasmid pGH-T58 of a human growth hormone fragment and an (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI fragment. In FIG. 14, p
GH-T58 is a fusion protein expression plasmid having a thrombin cleavage sequence.

Incidentally, similarly to the method for constructing pGH-T59, a human growth hormone expression plasmid pGH-L9 was used.
(Proc. Natl. Acad. Sci. USA., 81 , 5956, 19).
84) Gene fragment obtained by removing the latter third of the human growth hormone gene and SLPI subclone pUC-D
6 (WO89 / 06239) by restriction enzyme digestion and the like (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI fragment was ligated with a synthetic DNA linker (for example, a DNA fragment containing a gene encoding a thrombin cleavage sequence). Similarly, the expression type plasmid pGH-T58 can be obtained.

In the present invention, the gene encoding the recognition amino acid sequence of the fusion site that links the gene encoding human growth hormone or a part thereof has the same reading frame as that of the carrier protein human growth hormone or a part thereof. The fusion protein thus expressed is not particularly limited as long as it is an amino acid sequence which can be easily cleaved by treating the expressed fusion protein by the above-mentioned chemical method or biological method and can isolate the target protein. For example, (Asn-Gly) _n , (Asp-A
sp-Asp-Asp-Lys) _n , (Leu-Val)
-Pro-Arg) _n , (Val-Pro-Arg) _n
(N represents an integer of 1 to 10).

A synthetic DNA linker can also be inserted between the carrier protein and the genes encoding these recognition amino acid sequences to match the reading frame.

Further, (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI
As a gene fragment, this (Arg ⁵⁸ -Ala ¹⁰⁷⁾ SL
The gene corresponding to the PI polypeptide fragment, or its 2
Ten repetitive genes can also be used.

[0048] The fusion protein gene of the present invention can be made into an expression type gene by connecting it to the downstream of an appropriate promoter and SD sequence. Possible promoters include tryptophan operon promoter (trp promoter) and lactose operon promoter (la
c promoter), tac promoter, PL promoter,
The Ipp promoter, the T7 promoter and the like are preferable. In particular, the 5 ′ flanking sequence of pGH-L9 using the trp promoter and optimizing the interval between the SD sequence and the translation initiation signal ATG is preferable (Japanese Patent Application Laid-Open No. 60-234584).
No.).

The fusion protein gene expression type DNA for efficiently expressing the 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-linked peptide. It is preferable that the gene encoding the target protein or a part thereof and the translation termination codon be linked in this order. Such fusion protein gene expression type DNA
Is inserted into an appropriate plasmid, for example, pBR322 or a plasmid analogous 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, for example, prokaryotic cells such as Escherichia coli, Bacillus subtilis, and actinomycetes; mammalian cells such as CHO cells and COS cells; and insects such as silkworms. Cells include eukaryotic cells such as yeast cells. In particular, E. coli cells are preferred.

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

The transformed microorganism obtained in this way can be used in a manner known per se, for example in WO 89/06.
A method of culturing under the kind of medium and culturing conditions described in Japanese Patent No. 239, collecting the transformed microbial cells after culturing, crushing the cells, and isolating and purifying the fusion protein to obtain the fusion protein be able to.

The obtained fusion protein can be cleaved by treating it with the above-mentioned chemical method or biological method. If necessary, it is sulfonated to obtain a sulfonated molecule, and then the intact target protein is separated.・ Can be released.

In the method for producing the elastase inhibitory polypeptide of the present invention or its polymer, the 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 Can be implemented by the following method.

In the method for enzymatically treating SLPI of the present invention, the SLPI is a human leukocyte elastase inhibitory protein which is present in human saliva, nasal secretion, semen, uterine mucus, etc., eg, parotid gland secretion Origin S
LPI (RC Thompson et al., Proc. Natl. Acad. Sci. U
SA, 83 , 6692-6696, 1986;
-5011291); recombinant human SLPI obtained by genetic engineering using a gene encoding the amino acid sequence of natural human SLPI; or WO89 / 06239. Recombinant natural (Asn ⁵⁵ -Ala ¹⁰⁷ ) human SLPI fragments obtained by the described genetic engineering techniques, and the like.

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

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

[0058] Further, as a method of chemically synthesized using amino acids constituting the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 of the elastase inhibitory activity poly <br/> peptide production method of the present invention For example, the amino acid can be synthesized using such an amino acid as a raw material, which is a method known per se, using an automatic peptide synthesizer.

[0059] Elastase inhibitory activity polypeptide de a desired protein thus obtained is in itself known separation, suitably in combination separated from the reaction mixture in purification methods, it can be purified. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation,
Methods that mainly use differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and differences in charge, such as ion-exchange chromatography and ion-exchange high-performance liquid chromatography Method, method using specific affinity such as affinity chromatography, method using difference in hydrophobicity such as reversed phase high performance liquid chromatography, method using difference in isoelectric point such as isoelectric focusing And so on. When the obtained target protein or the like does not have a higher-order structure having biological activity, the target protein is converted into a higher-order having biological activity by using a known disulfide bond formation reaction. It can also be a structural protein.

When the target protein thus obtained forms a higher-order structure via a disulfide bond, for example,
Chance et al. (RE Chance et al., Peptides: 7th US Peptide Symposium Proceedings (DH Rich and
E. Gross), 721-728, Pierce Chemical Co.,
Rockford, IL. (1981)) or by using the method described in Japanese Patent Application No. 2-141523 already disclosed by the present inventors, a biological protein having a higher-order structure similar to that of a natural protein. Active protein.
In the method via the sulfonation method in the thrombin cleavage method, the target protein to be sulfonated is formed directly or after reduction to form an intramolecular disulfide bond to be a biologically active protein having the same higher-order structure as the natural protein. Can be.

The elastase inhibitory polypeptide of the present invention has the property of exhibiting elastase inhibitory activity and cathepsin G inhibitory activity, substantially not exhibiting trypsin inhibitory activity, and being derived from a natural form, Various diseases, for example, diseases caused by neutrophil hyperactivation, for example, elastase and / or cathepsin G, etc. A disease caused by a neutrophil-releasing protease, such as a therapeutic agent for inflammatory disease, platelet aggregation thrombosis, or post-ischemic reperfusion injury, especially 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 agent for RDS (respiratory distress syndrome), asthma, and interstitial pneumonia.

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

The pharmaceutical preparations and therapeutic agents of the present invention can be administered transtracheally or orally, or parenterally such as intravenously, subcutaneously, intramuscularly, transdermally, or rectally.

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

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

For tableting, for example, excipients such as lactose, starch, and crystalline cellulose; binders such as carboxymethyl cellulose, gelatin, and polyvinylpyrrolidone; disintegrants such as sodium alginate, starch, and crystalline cellulose are used. And can be molded by an ordinary method.

Similarly, pills, powders, and granules can be formed by an ordinary method using the above-mentioned excipients and the like.

Solutions and suspensions can be formed by a conventional method using, for example, glycerin esters such as triacetin and alcohols such as ethanol. Capsules can be formed by filling granules, powders, solutions or the like 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. Physiological saline or the like is used as the aqueous solution. The water-insoluble solution includes, for example, propylene glycol, polyethylene glycol, olive oil, ethyl oleate, and the like.
Stabilizers and the like are added. The injection is sterilized by appropriate treatment such as filtration through a bacteria removal filter and blending of a bactericide.

Examples of dosage forms for transdermal administration include, for example, ointments,
Creams and the like can be mentioned, and ointments are molded by a usual method using a fatty oil such as castor oil and olive oil; vaseline and the like, and creams are a fatty oil; emulsifiers such as diethylene glycol and sorbitan monofatty acid ester by an ordinary method. be able to.

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

The dosage of the polypeptide of the present invention varies depending on the administration route, age of the patient, sex, and the degree of the disease.
Usually, the dose is about 1 to 5000 mg / time, and the number of doses is usually 1
３3 times / day, and it is preferable to adjust the preparation so as to satisfy such conditions.

[0073]

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

The various genetic engineering techniques employed in Reference Examples and Examples were carried out according to the methods described in WO 89/06239, pp. 24-26.

[0075]

Reference Example 1 Preparation of (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI (Asn ⁵⁵ -Ala ¹⁰⁷ )
n ⁵⁵ -Ala ¹⁰⁷ ) SLPI was produced.

That is, (Asn ⁵⁵ -Ala ¹⁰⁷ ) SL
A DNA fragment encoding PI was chemically synthesized using appropriate codons. Leu that can be cut with thrombin
Via a DNA sequence encoding Val-Pro-Arg, the human growth hormone gene was transformed into (Asn ⁵⁵ -Al
a ¹⁰⁷ ) fused to SLPI. The thus obtained expression vector (plasmid pGH-TE) was used in E. coli. col
iHB101. Culturing the transformed cells (E. coli HB 101 strain (pGH-TE)), to give a capsule combined, and purified by thrombin treatment and S-S reconfiguration further chromatography, the objective (Asn ⁵⁵ -Al
a ¹⁰⁷ ) SLPI was obtained. The obtained (Asn ⁵⁵ -Ala)
¹⁰⁷ ) SLPI is based on its Cys ⁶⁴ -Cys ⁹³ , Cys ⁹²
-Cys ^80, Cys ⁷¹ -Cys ^97, and Cys ⁸⁶ -Cy
the Cys residues of s ¹⁰¹ are those that form four disulfide bonds to each other was confirmed by Mass spectrum and two-dimensional NMR.

The Escherichia coli HB101 strain (pGH
-TE) is a microfabrication researcher Yoko 2168 (FERM BP-
2168) in 1992
It was deposited internationally 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). WO89 / 06
In the same manner as in the example of JP-A-239, the human growth hormone gene was converted to SL via a DNA sequence encoding Leu-Val-Pro-Arg, which can be cut with thrombin.
The DNA encoding PI was fused. The thus obtained expression vector (plasmid pGH-SLPI) was used in E. coli. coli HB101. Transformed cells (Escherichia coli HB101 strain (pGH-SLPI))
To obtain a conjugate, purified by thrombin treatment, SS reconstitution, and chromatography to obtain the desired S
LPI was obtained. The inhibition constant (Ki) of the obtained SLPI against elastase was 2.5 × 10 ⁻¹⁰ M, which was equivalent to the activity of SLPI isolated from nature (Japanese Patent Application Laid-Open No. Sho 62-5).
01291).

[0079]

Example 1 Identification of SLPI Degraded Product by Normal Human Saliva (1) Normal human saliva was subjected to ultrafiltration with a 0.22 μm filter (Mirex GS, Millipore), and the SLPI obtained in Reference Example 2 was purified. It was dissolved at a concentration of 1 mg / ml and kept at 37 ° C. for 30 days. Saliva after filtration and SLPI were added to physiological saline (Fisizalz, Fuso Pharmaceutical) at 1 mg / ml.
Was dissolved at a concentration of 37, and used as a control sample.
The temperature was kept at 30 ° C until 30 days.

After each of these specimens was collected over time, Tr
is-HCl buffer (pH 6.8), SDS, 2-
Mercaptoethanol and glycerol were added to a final concentration of 60 mM, 2 w / w%, 4 w / w%, and 10 w / w%, respectively, and SDS-PAGE (UK Laemmli, Nature,
227 , 680, 1970). Figure 1 shows the results.
Shown in The gel after electrophoresis was stained with Coomassie brilliant blue. In the figure, lane No. Two bands a (molecular weight: about 13,000) indicate SLPI bands.

As is clear from FIG.
Since the day, the molecular weight has reached 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). In addition, the molecular weight of about 13,000 expected for SLPI
It was confirmed that band a decreased as the heat retention time increased, while bands b, c and e increased. In addition, SLPI in the physiological saline, despite the elapse of the heat retention time,
A band having a molecular weight of about 13,000 was maintained (lane N
o. 9). In addition, SDS-P at 37 ° C for 30 days
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 subjected to Milli Blot-SDE
Using a device (Millipore), the membrane (Im
mobilon, Millipore). After the transfer, the membrane was stained with 0.1 w / w% Coomer Seabre / 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 to an Applied Biosystems protein sequencer (Applied Biosystems; 47).
0A), the PTH-amino acid was cleaved from the N-terminus, and the PTH analyzer (Applied Biosystems, 1
20A), and the amino acid sequence up to the tenth from the N-terminus was determined. Table 1 shows the results. In addition,
Band e had two N-terminal amino acid sequences.

[0083]

[Table 1]

From Table 1, the bands b and c are the TPI of SLPI.
amino acid sequence from r ^21, also band d is the amino acid sequence from Arg ^59, band e was exactly matches 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- Arg ⁵⁹ , Leu ⁷²
-Between Met ⁷³ and between Met ^{73 and} Leu ⁷⁴ .

From the N-terminal amino acid sequences of bands b, c, d, and e (Table 1) and the molecular weight (FIG. 1), it was confirmed that each polypeptide was a polypeptide as shown in Table 2 below. Was.

[0086]

[Table 2]

[0087]

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

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

[0089]

[Table 3]

From the results, 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
Conversion to I was confirmed.

[0092]

EXAMPLE 3 polypeptides 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 to an automatic peptide synthesizer (Applied Biosystems, 4
31A) and reverse phase chromatography (YM
(C-PackODS-AP, YMC). Since the obtained peptide has a non-natural type 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 intramolecular disulfide bond by a refolding reaction (Arg ⁵⁹ -Ala
¹⁰⁷ ) SLPI was obtained. That is, 20 mg of the polypeptide of SEQ ID NO: 2 synthesized by an automatic peptide synthesizer was added to 0.1 mg
2. 25M Tris Buffer (pH 8.2);
After dissolving in 5 ml of 5 M urea, 0.35 M Na ₂ SO ₃ and 0.06 M sodium tetrathionate in 20 ml and reacting at room temperature for 2 hours, the reaction solution was subjected to reverse phase chromatography (Prot.
ein C ₄ , VYDAC) and freeze-dried (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI sulfonated derivative 18
mg was obtained.

The refolding reaction and purification were carried out in the same manner 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 reacted at 4 ° C. for 7 days. Next, S-Seph
arose (Pharmacia) ion exchange chromatography, reverse phase chromatography (Protein C ₄ , VYD)
AC Co.), and an oligomer by-produced by refolding was separated. Then freeze-dried, (Arg ⁵⁹ -A
la ¹⁰⁷ ) 7.8 mg of SLPI were obtained. The obtained polypeptide was subjected to reverse phase chromatography (Protein C ₄ , VY
(DAC), it was eluted as a single peak, and it was confirmed that it was a single substance. The elution pattern is shown in FIG.

[0095]

Example 4 Elastase of (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI and
Measurement of trypsin inhibitory activity The measurement method is described below.

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

[0097]

[Table 4]

(2) Method 120 μl was placed in the well of a 96-well microplate for ELISA.
Of buffer solution, 20 μl of the sample solution of (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI obtained in Example 3 was added, and then 20 μl of the enzyme solution was added.
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 a color, and the absorbance was measured at 405 nm. Using SLPI as a control, the concentration of the solution was changed and the inhibitory activity was measured to determine the inhibitory protein concentration (IC ₅₀ value) showing a 50% inhibitory concentration. Table 5 shows the results.

The oligomer by-produced in Example 3 measured in a similar manner had an IC ₅₀ value of 3.0 × 10 ⁻⁷ M for elastase.

[0100]

[Table 5]

[0101]

Example 5 Cathepsin G Inhibition of (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI
Measurement of activity 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 of buffer (0.1 M HEPES, 1.0 M Na
Cl, 0.1% PEG 6000 pH 7.5)
Next, 20 μl of the product obtained in Example 3 (Arg ⁵⁹ -Ala)
¹⁰⁷ ) A sample solution of SLPI 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 of the substrate solution (Suc-Phe-Pro-Phe-pN
A; a final concentration of 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 Showed inhibitory activity.

[0102]

EXAMPLE 6 Human growth hormone fragment polypeptide and (Arg ⁵⁹ -Al
a ¹⁰⁷ ) Expression plasmid for fusion protein of SLPI polypeptide
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 cut with restriction enzymes BglII and SalI (Takara Shuzo), separated by agarose gel electrophoresis, and about 4.7 kbp DNA from the gel.
The fragment was recovered using GENECLEAN II KIT (manufactured by BIO101).

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

On the other hand, D containing the thrombin cleavage sequence gene
The NA fragment (FIG. 4) was synthesized using a fully automatic synthesizer (Applied Biosystems, Inc. model 392). The synthetic DNA fragment was MEGALABEL
After phosphorylation of the 5 'end using ^TH (Takara Shuzo) in the presence of 1 mM ATP, annealing with a synthetic DNA fragment was performed to obtain a double-stranded DNA. The annealing reaction was left at room temperature after heating at 90 ° C. for 5 minutes.

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

(2) The cross-linked peptide is enterokinase-cleaved
Expression plasmid pGH-T59 (FIG. 5) having a cleavage sequence
Fragment (Fig. 6) as primer
Rase chain reaction; PCR method (Saiki et al., Science,23
9, 487-491, 1985).
Again to the synthetic DNA fragment and (FIG. 6)
BglII (Takara) after amplification by PCR
) To obtain a fragment of about 115 bp.

The conditions of the PCR method were as follows: after denaturation at 93 ° C. for 5 minutes, one cycle was performed at 93 ° C. for 1.5 minutes
C., 2 minutes, 72 ° C., 2 minutes, 30 cycles,
Finally, the reaction was stopped at 72 ° C. for 7 minutes.

On the other hand, after cutting pGH-T59 with BglII (Takara Shuzo), a BglII-BglII fragment of about 4.6 kbp was separated by agarose gel electrophoresis.
It was collected using EAN II (manufactured by BIO101). Using a Bacterial Alkaline Phosphatase (Takara Shuzo), the approximately 4.6 kbp BglII-BglII fragment was subjected to a 5′-terminal dephosphorylation reaction.

About 4.6 kbp dephosphorylated BglII
-BglII fragment and about 115 bp amplified by PCR
After performing a ligation reaction using the ligation kit (Takara Shuzo), the BglII-BglII fragment of coli HB1
The cells were introduced into 01 competent cells (Takara Shuzo) and cultured on an LB agar medium containing ampicillin to obtain a transformant (HB / GH-E59) containing the desired fusion protein expression plasmid.

[0110] Plasmid DNA was extracted from the transformant, QIAGEN purified (manufactured by BIO101 companies), with the human growth hormone fragment polypeptide and enterokinase fragment sequence gene (Arg ⁵⁹ -Ala ¹⁰⁷⁾ SLPI
Plasmid fusion protein expression plasmid pGH-E59
(FIG. 7).

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

[0112]

Example 7 Expression of fusion protein gene Expression plasmid pG of the fusion protein gene obtained in Example 6
E. coli HB101 strain containing HT59 was
% Glucose, 3 mg / ml yeast extract (DIFCO), modified M9 medium containing 20 mg / ml casamino acid (DIFCO) (1% (NH ₄ ) ₂ HPO ₄ -0.3%
After autoclaving K ₂ SO ₄ -0.3% NaCl), MgSO ₄ and CaCl ₂ were added to a final concentration of 1%.
The cells were inoculated into 500 ml of a culture solution aseptically added to a concentration of mM and cultured overnight.

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

The washed cells were suspended in a 0.5 M Tris buffer containing lysozyme (1/200 of the wet weight of the cells) and EDTA · 3Na (1/20 of the wet weight of the cells), and sonicated. Generator (CELL DISRU, manufactured by BRANSON)
After disrupting the cells using PTOR 900, the cells are centrifuged, and the desired fusion protein is included in the precipitate (inclusion bod).
y).

E. coli H containing pGH-T59
B101 strain (HB / GH-T59)
No. 63 (FERM BP-3863) and deposited on May 20, 1992 at the Research Institute of Microbial Industry of the National Institute of Advanced Industrial Science and Technology.

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

[0117]

Embodiment 8Sulfonation of cysteine residues in 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
And solubilized, and the final concentration is 0.7 M
Sodium sulfite (Wako Pure Chemicals)
The reaction was performed at 5 ° C. for 20 minutes. Further, the final concentration is 0.1 M.
Sodium 4-thionate (Sigma)
And reacted at 45 ° C. for 20 minutes. 10 mM Tris-ba
Buffer (pH 8.0), and add 1 liter of the reaction solution.
And then ultrafiltration until the volume reaches 200 ml
Filter (FILTRON) 5 times
I returned. Thus, the obtained sulfonated fusion protein solution
300 parts for the liquid (the part that did not pass through the ultrafiltration membrane)
Add 0 U of bovine thrombin (Mochida Pharmaceutical) and add 3 U at 37 ° C.
Allowed to react for hours. Then, by ultrafiltration, 300K
Contained in the solution that passed through the filter (Arg⁵⁹-Ala
¹⁰⁷) SLPI sulfonated derivative was subjected to reverse phase HPLC (VY
DAC; Protein C_Four) And lyophilized. Profit
(Arg ⁵⁹-Ala¹⁰⁷) SLPI sulfonation induction
The N-terminal amino acid sequence of the body
(Applied Biosystems 477A).

[0118]

Example 9 Activity of (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI sulfonated derivative
Refolding of sexually molecules (disulfide reaction) obtained in Example 8 (Arg ⁵⁹ -Ala ¹⁰⁷⁾ SLPI
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), reacted at 4 ° C. for 2 days, and then subjected to S-Sepharose (Pharmacia) ion exchange chromatography. The elution pattern was shown in FIG. Show.
The desired fraction eluted with 0.3 M NaCl was collected on a reverse phase HP
After further purification by LC (VYDAC, Protein C ₄ ), it was freeze-dried and activated (Arg ⁵⁹ -Ala ¹⁰⁷ ) SL
PI was obtained.

[0119]

[Example 10] (Arg ⁵⁹ -Ala ¹⁰⁷⁾ serine protease over of SLPI
(1) Measurement method is shown below.

(1) Reagent solution Buffer: 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 so as to have a concentration 9 times the concentration shown in Table 6. A. B. Human sputum-derived polymorphonuclear leukocyte elastase (EPC, Funakoshi Pharmaceutical) Bovine spleen trypsin (Sigma) C.I. Bovine spleen chymotrypsin (Sigma) D. Pig spleen elastase (manufactured by Sigma) Human-derived cathepsin G (manufactured by EPC, Funakoshi Pharmaceutical) Substrate solution: Enzyme A. B. C. D. For each E, the following substrates a, b, c, d, and e were added in a concentration of 9 times the concentration shown in Table 6 in DMSO: buffer solution (1:
9) Dissolve in solution (DMSO final concentration is 10 in buffer)
%). 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 (Hansui Chemicals) e. Suc-Phe-Pro-Phe-pNA (Bac
hem)

[0121]

[Table 6]

(2) Method 120 μl in the well of a 96-well microplate for ELISA
Of buffer solution, 20 μl of the sample solution of (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI obtained in Example 9 was added, and then 20 μl of the enzyme solution was added.
The reaction was performed at 7 ° C. for 10 minutes. Next, 20 μl of the substrate solution was added, the color was developed at 37 ° C., and the absorbance was measured at 405 nm.

Using SLPI as a control, the concentration of the solution was varied, and the inhibitory activity was measured to determine the inhibitory protein concentration (IC ₅₀ value) showing a 50% inhibitory concentration. The results are shown in Table 7.

[0124]

[Table 7]

[0125]

[Example 11] (Arg ⁵⁹ -Ala ¹⁰⁷⁾ serine protease over of SLPI
(2) Measurement of (Arg ⁵⁹ -Ala ) in the following four enzymes
¹⁰⁷ ) The inhibitory protein concentration (IC ₅₀ ) value showing 50% inhibition of SLPI and SLPI as a control was determined. F. Thrombin derived from human plasma (manufactured by Sigma) G. Human plasma-derived plasmin (manufactured by Sigma) Human plasma-derived kallikrein (protogen, Funakoshi) I. The method for measuring pig-derived FXa (first chemical) 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, pH7.
8 Enzyme concentration; Table 8 shows the final concentration of each enzyme.

[0128]

[Table 8]

Substrate solution: enzyme F. G. FIG. H. Substrates f, g, h and i shown below were used for each of I, and their final concentrations are shown in Table 8. f. HDD-Phe-Pip-Arg-pNA (Kabi V
itrum company) g. HDVal-Leu-Lys-pNA (Kabi V
itrum company) h. HDD-Pro-Phe-Arg-pNA (Kabi V
itrum company)

[0130]

Embedded image

(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.
Incubated for a minute. Next, a substrate solution is added and color is developed at 37 ° C.
The absorbance was measured at 405 nm.

As a control, SLPI was used.

(3) Results In the case of (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI and SLPI at a final concentration of 1 × 10 ⁻⁵ M or less, no inhibitory activity was observed for all of the above four enzymes.

[0134]

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

(1) Reagent solution Buffer: 0.1 M N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES)
1.0 M NaCl 0.1% polyethylene glycol (pH 7.5) Enzyme solution: Dissolve each of the following enzymes in the above buffer so that the concentration is 9 times the concentration shown in Table 9. (A) Human sputum-derived polymorphonuclear leukocyte elastase (EPC
(B) Bovine spleen trypsin (manufactured by Sigma) Substrate solution: Substrates of the following (d) and (e) were added to each of the above enzymes (a) and (b) at the concentrations shown in Table 9 respectively. In (d), the buffer is dissolved 1: 9 in DMSO, and (e) is dissolved in DMSO to obtain 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 in the well of a 96-well microplate for ELISA
Of buffer solution, 20 μl of the sample solution of (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI obtained in Example 9 was added, and then 20 μl of the enzyme solution was added.
The reaction was performed at 5 ° C. for 1 hour. Next, 20 μl of the substrate solution was added, the color was developed at 25 ° C., and the absorbance was measured at 405 nm. SLPI was used as a control.

From the results, the method of Henderson et al.
son, PJF (1972), Biochem. J. 127 , 321-3
In accordance with the present invention with ^{33) (Arg 59 -Ala 107)} SL
The inhibition constant (Ki) value of PI was determined. The results are shown in Table 10.

From Table 10, the active form (A) obtained in Example 9 was obtained.
rg ⁵⁹ -Ala ¹⁰⁷⁾ relative activity ratio of the trypsin inhibitory activity against elastase inhibitory activity of SLPI inhibition constants for (inhibition constants ki (T for trypsin) / elastase ki (E)) 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 with polymorphonuclear leukocyte elastase from human sputum
Of human sputum-derived polymorphonuclear leukocyte elastase (manufactured by EPC,
Funakoshi Pharmaceutical Co., Ltd.) and SLPI were each added to a buffer solution (0.
1M HEPES, 1.0M NaCl, 0.1% PE
G6000, pH 7.5)
An elastase solution was added to the I solution, and the mixture was kept at 37 ° C. After collecting each of these specimens in a timely manner, Tris-HC
1 buffer (pH 6.8), SDS, 2-mercaptoethanol, glycerol, and Coomassie brilliant blue at a final concentration of 50 mM, 4 w / v%, 2 v / v, respectively.
%, 12 v / v%, 0.01 w / v%, and T
SDS-polyacrylamide gel electrophoresis (Schagger,
H., Anal. Biochem. 166 , 368, 1987). The gel after electrophoresis is Coomassie Brilliant Blue G
50 v / v% methanol / 10 v / v stained with -250
% Acetic acid.

(1) To 100 μM of SLPI, elastase was added at a final concentration of 200 μM, and the elastase was added for 3 hours until 192 hours.
The result of keeping the temperature at 7 ° C. is shown in FIG. Samples were taken over time. SLPI (SDS) of lane 2 band a in FIG.
-The molecular weight is about 14000 on PAGE)
It decreased remarkably after 5 minutes. Accordingly, band b
(Molecular weight of about 11,000) appeared from the 45th minute and was observed until the 6th hour. 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 by a factor of 2 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 to μM. Band e of lane 2 in FIG.
(Molecular weight of about 14,000) indicates a band of SLPI. As is clear from FIG. 10, SLPI 50 μM
On the other hand, when elastase was at a final concentration of 100 μM or more, remarkable degradation of SLPI of band e was observed. Band f (molecular weight: about 11,000) and band g (molecular weight: about 5,500) are clearly novel bands, and appeared as the band e disappeared, indicating that they were SLPI degradation products.

(3) Next, the polypeptide in the polyacrylamide gel thus obtained was subjected to Milli Blot-SDE
Using a device (Millipore), the membrane (Im
mobilon-P ^SQ , Millipore). After transfer, the membrane is washed with 0.1 w / v% Coomassie blue / 40 v / v
Stained with 1% methanol / 1% v / v acetic acid and washed with water. After air-drying, cut out the target band, and use Applied Biosystems
Protein sequencer (Applied Biosystems; 4
77A), the amino acid is cleaved from the N-terminus, and after PTH conversion, a PTH-amino acid analyzer (Applied Biosystems)
ms, 120A) to determine the N-terminal amino acid sequence. Table 11 shows the results.

[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 sites of SLPI in human sputum-derived elastase were between Ser ^{15 and} Ala ¹⁶ and between Thr ^{57 and} Arg ⁵⁸ .

Further, based on the N-terminal amino acid sequences of bands b and f and bands c and g (Table 11) and the decomposition pattern and 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: Polymorphonuclear leukocyte elastase from normal human saliva and human sputum
Simultaneous processing of SLPI by (1) SLPI and human sputum from Polymorphonuclear leukocyte elastase (EPC Co., Funakoshi Pharmaceutical) a 0.22μm filter (Millex GS, Millipore) at normal human saliva ultrafiltration, Each was dissolved at a final concentration of 50 μM. This mixed solution was kept 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: about 14,000) shown in lane 2 band I was incubated at 37 ° C. in saliva in which elastase was present at an equal concentration,
At 96 hours, Band J with a molecular weight of about 5,500 newly appeared.

(2) Next, the band J was electrically transferred to a membrane in the same manner as in Example 1, and the N-terminal amino acid sequence was analyzed. Table 13 shows the results. These results, N-terminal sequence of the band J is in complete agreement with SLPI amino acid sequence after Arg ^58.

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

[0152]

[Table 13]

[0153]

Example 15: Polymorphonuclear leukocyte elastase from normal human saliva and human sputum
(Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI simultaneous treatment with (A) 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 μM, respectively.
The mixture was dissolved in 0 μM, and the mixed solution was kept at 37 ° C.
This sample was collected over time and analyzed by SDS-PAGE.

FIG. 12 shows the result. (Asn ⁵⁵ -A
la ¹⁰⁷⁾ SLPI (molecular weight of about 5,800, band L)
Was treated simultaneously with normal human saliva and elastase, whereby band M having a slightly lower molecular weight than band L was 3
It appeared again at 12 hours at 7 ° C. The band L gradually decreased with the extension of the heat retention time, and after 48 hours, almost only 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. Table 14 shows the results.

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

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

[0158]

[Table 14]

[0159]

Example 16 (Asn) by polymorphonuclear leukocyte elastase derived from human sputum
^55- Ala ¹⁰⁷ ) SLPI treatment In the same manner as in Example 13, (A)
sn ⁵⁵ -Ala ¹⁰⁷ ) with a final concentration of 200 against SLPI
The cells were 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, it was electrically transferred to a filter, and a band corresponding to a molecular weight of about 5,500 was cut out.
The N-terminal amino acid sequence was analyzed. Table 15 shows the results.

From these results, it was found that Eras
(Asn⁵⁵-Ala ¹⁰⁷) SLPI N
The terminal amino acid sequence is Arg⁵⁸Subsequent SLPI amino acids
Converted to an array.

As described above, (Asn ⁵⁵ -Ala ¹⁰⁷ ) SL
PI is expressed by Thr ^57- by elastase derived from human sputum.
Cleaved between Arg ⁵⁸ and all (Arg ⁵⁸ -Al
a ¹⁰⁷ ) Conversion to SLPI was confirmed.

[0163]

[Table 15]

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

[0165]

Example 17 Human Growth Hormone Fragment Polypeptide (Arg ⁵⁸ -Ala)
¹⁰⁷ ) Expression plasmid for fusion protein of SLPI polypeptide
And preparation of a transformant thereof (Met ^-1 Phe ¹ -Phe ¹³⁹ ) A fusion protein expression plasmid pGH- of a human growth hormone fragment polypeptide and an enterokinase cleavage sequence gene (Arg ⁵⁹ -Ala ¹⁰⁷ ) SLPI polypeptide Polymerase chain reaction: PCR method (Saiki,
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 is again synthesized with a synthetic DNA fragment.
After amplification by PCR using (FIG. 13) as a primer, the fragment was cut with BglII (Takara Shuzo) to obtain a fragment of about 120 bp. Note that pGH-E59 was obtained in Example 6.

The conditions for the PCR method were as follows: after denaturation at 93 ° C. for 5 minutes, one cycle was performed at 93 ° C. for 1.5 minutes, 57 ° C. for 2 minutes, and 72 ° C. for 2 minutes, and finally 30 cycles
The reaction was stopped at 7 ° C. for 7 minutes.

On the other hand, after pGH-E59 was cut with BglII (Takara Shuzo), a BglII-BglII fragment of about 4.6 kbp was separated by agarose gel electrophoresis.
It was collected using LEAN II (manufactured by BIO101). Using a Bacterial Alkaline Phosphatase (Takara Shuzo), the approximately 4.6 kbp BglII-BglII fragment was subjected to a 5′-terminal dephosphorylation reaction.

About 4.6 kbp of dephosphorylated BglII
-BglII fragment and about 115 bp amplified by PCR
After performing a ligation reaction using the ligation kit (Takara Shuzo), the BglII-BglII fragment of coli HB1
The cells were introduced into 01 competent cells (Takara Shuzo) and cultured on an LB agar medium containing ampicillin to obtain a transformant (HB / GH-T58) containing the target fusion protein expression plasmid. Extract plasmid DNA from 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 using the plasmid DNA as a template, the synthetic DNA as a primer, and the dye terminator kit.
(Hitachi), and directly confirmed by a fluorescent automatic DNA sequencer SQ3000 manufactured by Hitachi.

[0170]

Example 18 Expression of Fusion Protein Gene Fusion protein gene expression plasmid p obtained in Example 17
E. coli HB101 strain containing GH-T58 was
5% glucose, 3 mg / ml yeast extract (DIFCO
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 sterilized by autoclave, and then inoculated into 500 ml of a culture solution to which MgSO ₄ and CaCl ₂ were aseptically added to a final concentration of 1 mM, and cultured overnight.

The overnight culture was used in the same manner as the culture 1
To 0 liter, OD660 was added to 0.2, and jar fermenter culture was performed at 37 ° C. O
When D660 became 20, 3-β-indoleacrylic acid was added to the culture solution to a final concentration of 150 μg / ml, and the culture was continued at 37 ° C. for 4 hours. Thereafter, E. coli cells were collected by centrifugation, and washed with 0.5 M Tris buffer (pH 8.0).

The washed cells were suspended in a 0.5 M Tris buffer containing lysozyme (1/200 of the wet weight of the cells) and EDTA · 3Na (1/20 of the wet weight of the cells). Generator (CELL DISRU, manufactured by BRANSON)
After disrupting the cells using PTOR 900, the cells are centrifuged, and the desired fusion protein is included in the precipitate (inclusion bod).
y).

E. coli H containing pGH-T58
B101 strain (HB / GH-T58)
No. 62 (FERM BP-3862) and deposited on May 20, 1992 at the Research Institute of Microbial Industry of the National Institute of Advanced Industrial Science and Technology.

[0174]

Example 19 Sulfonation of Cysteine Residue of Fusion Protein and Sulfonated Fusion Protein
Cleavage reaction with white thrombin 25 g of the fusion protein obtained in Example 18 was suspended in 200 ml of 0.5 M Tris buffer (pH 8.0).
240 g of urea was added to solubilize, and the final concentration was 0.7
Sodium sulfite (Wako Pure Chemical Industries, Ltd.) was added so as to obtain M and reacted at 45 ° C. for 20 minutes. Further, sodium 4-thionate (Sigma) was added to a final concentration of 0.1 M, and the mixture was reacted at 45 ° C. for 20 minutes. After adding 10 mM Tris buffer (pH 8.0) to make the reaction solution 1 liter, the operation of ultrafiltration (Fujifilter; FILTRON) was repeated 5 times until the volume became 200 ml. The resulting sulfonated fusion protein solution (the portion that did not pass through the ultrafiltration membrane) was treated with 300
Add 0 U bovine thrombin (Mochida Pharmaceutical) and add 3 U at 37 ° C.
Allowed to react for hours. Then, the solution was passed through a 300K filter by ultrafiltration (Arg ⁵⁸ -Ala).
¹⁰⁷ ) The SLPI sulfonated derivative was subjected to reverse phase HPLC (VY
Purified by DAC; Plotein C4) and freeze-dried. The resulting (Arg ⁵⁸ -Ala ¹⁰⁷⁾ N-terminal amino acid sequence of SLPI sulfonated derivative was determined by Protein Sequencer (Applied Biosystems).

[0175]

Example 20 Activity of (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI sulfonated derivative
Obtained in Refolding (disulfide reaction) Example 19 to sexual molecules (Arg ⁵⁸ -Ala ¹⁰⁷⁾ SLP
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), reacted at 4 ° C. for 2 days, and then subjected to S-Sepharose (Pharmacia) ion-exchange chromatography. Show.
The desired fraction eluted with 0.3 M NaCl was separated by reverse phase HPL.
After further purification with C (VYDAC, Protein C4)
Lyophilized, activated (Arg ⁵⁸ -Ala ¹⁰⁷⁾ SLPI
I got

[0176]

[Example 21] (Asn ⁵⁸ -Ala ¹⁰⁷⁾ serine protease over of SLPI
(1) Measurement method is shown below.

(1) Reagent solution Buffer: 0.1 M- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES) 0.5
M NaCl, pH 7.5 Enzyme concentration: The following enzymes are dissolved in the above buffer so as to have a concentration 9 times the concentration shown in Table 16. A. B. Human sputum-derived polymorphonuclear leukocyte elastase (manufactured by EPC, Nafcosi Pharmaceutical) Bovine spleen trypsin (Sigma) C.I. Bovine spleen chymotrypsin (Sigma) D. Pig spleen elastase (manufactured by Sigma) Human-derived cathepsin G (manufactured by EPC, Funakoshi Pharmaceutical Co.) Substrate solution: Substrates of the following a, b, c, d, and e for each of the above enzymes A, B, C, D, and E have the concentrations shown in Table 16 respectively. DMSO: The above buffer solution (1:
9) Dissolve in solution (DMSO final concentration is 10 in buffer)
%). 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 (Hansui Chemicals) e. Suc-Phe-Pro-Phe-pNA (Bac
hem)

[0178]

[Table 16]

(2) Method 120 μl in the well of a 96-well microplate for ELISA
Of buffer solution, 20 μl of the sample solution of (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI obtained in Example 20, and then 20 μl of the enzyme solution are added.
The reaction was performed at 7 ° C. for 10 minutes. Next, 20 μl of the substrate solution was added, the color was developed at 37 ° C., and the absorbance was measured at 405 nm.

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

[0181]

[Table 17]

[0182]

[Example 22] (Arg ⁵⁸ -Ala ¹⁰⁷⁾ serine protease over of SLPI
(2) Measurement of (Arg ⁵⁸ -Ala ) in the following four enzymes
¹⁰⁷ ) The inhibitory protein concentration (IC ₅₀ ) value showing 50% inhibition of SLPI and SLPI as a control was determined. F. Thrombin derived from human plasma (manufactured by Sigma) G. Human plasma-derived plasmin (manufactured by Sigma) Human plasma-derived kallikrein (protogen, Funakoshi) I. The method for measuring pig-derived FXa (first chemical) 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, pH7.
8 Enzyme concentration; 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. HDD-Phe-Pip-Arg-pNA (Kabi V
itrum company) g. HDVal-Leu-Lys-pNA (Kabi V
itrum company) h. HDD-Pro-Phe-Arg-pNA (Kabi V
itrum company)

[0184]

Embedded image

[0185]

[Table 18]

(2) Method Example 20 was inserted into the holes of a 96-well microplate for ELISA.
The sample solution of (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI obtained in the above was added, and then the enzyme solution was added.
It was kept at 7 ° C. for 10 minutes. Then add the substrate solution and add
And the absorbance was measured at 405 nm.

[0187] 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 Inhibitor constant of (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI (K
i) The method of measuring the value is shown below.

(1) Reagent solution buffer: 0.1 M 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 so that the concentration is 9 times the concentration shown in Table 19. (A) Human sputum-derived polymorphonuclear leukocyte elastase (EPC
(B) Bovine spleen trypsin (manufactured by Sigma) Substrate solution: Substrates of the following (d) and (e) were added to each of the above enzymes (a) and (b) at the concentrations shown in Table 19, respectively. In (d), the buffer is dissolved 1: 9 in DMSO, and (e) is dissolved in DMSO to obtain a substrate solution. (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 in the well of a 96-well microplate for ELISA
Of buffer solution, then add 20 μl of the sample solution of (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI obtained in Example 20, then add 20 μl of enzyme solution and add 2 μl of this mixture.
The reaction was performed at 5 ° C. for 1 hour. Next, 20 μl of the substrate solution was added, the color was developed at 25 ° C., and the absorbance was measured at 405 nm. SLPI was used as a control.

From the results, the method of Henderson et al.
son, PJF (1972), Biochem. J. 127 , 32
(Arg ⁵⁸ -Al) of the present invention in accordance with 1-333).
a ¹⁰⁷ ) 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 (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI obtained in Example 20 (inhibition constant ki (T) for trypsin / inhibition constant ki for elastase ki) (E)) was about 12,000. That is, (Arg ⁵⁸ -Ala ¹⁰⁷ ) SLPI did not substantially exhibit 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 the drawings]

FIG. 1 shows an SDS-PAGE of limited degradation products when SLPI was treated 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), b represents a limited degraded product (Tyr ²¹ -Ala ¹⁰⁷ ) SLPI, and c represents a limited degraded product ((Tyr ²¹ -Leu ⁷² ) or (Tyr ²¹ -Met ⁷³ )).
SLPI), d is the polypeptide of the present invention (MW about 5,5
00), e is ((Met ⁷³ -Ala ¹⁰⁷ )) or (Leu
⁷⁴ shows the band of -Ala ¹⁰⁷⁾ SLPI).

FIG. 2 shows (Asn ⁵⁵ -Ala ¹⁰⁷ ) S in Example 2.
SD of limited degradation product 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), and g indicates the band of the polypeptide of the present invention (MW about 5,500).

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

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

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

FIG. 6 shows a synthetic DNA fragment (,,) used in Example 6 (2).

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

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

FIG. 9 shows SDS-PA when SLPI was treated with twice the amount of human polymorphonuclear leukocyte elastase in Example 13.
GE is shown. In the figure, the band of the line indicated by a is SL
The band of PI (MW about 14,000) is shown, b shows the band of the limited digest (Ala ¹⁶ -Ala ¹⁰⁷ ) SLPI, and similarly, c shows the band of the polypeptide of the present invention (MW of about 5,500). Also, a group of bands d indicates elastase.

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

FIG. 11 shows SDS-PAGE of a limited degradation product when SLPI is simultaneously treated 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 a band of the polypeptide of the present invention (MW: about 5,500). Also,
A group of bands K indicates elastase.

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

FIG. 13 shows a synthetic DNA fragment in Example 17.

FIG. 14 is a graph showing (Arg ⁵⁸ —Al
a ¹⁰⁷ ) Shows construction of SLPI polypeptide fusion protein expression plasmid pGH-T58.

FIG. 15 shows the active form (Arg ⁵⁸ -Al
a ¹⁰⁷⁾ shows the elution pattern by S-Sepharose ion exchange chromatography SLPI.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 19/00 C12P 21/02 C C12N 1/21 A61K 37/64 ABE C12P 21/06 ABN // C12P 21/02 ACB (C12N 1/21 (C12R 1:19) (C12P 21/02 C12R 1:19) Accession number of microorganisms FERM BP-3862 Accession number of microorganisms FERM BP-3863 (72) Inventor Tetsuya Suga 4-2-2 Asahioka, Hino-shi, Tokyo Tei Inside the Tokyo Research Center (72) Inventor Akiko Takeuchi 4-2-2 Asahigaoka, Hino-shi, Tokyo Teijin Inside the Tokyo Research Center (72) Inventor Yoichi Matsumoto 4-2-2 Asahigaoka, Hino-shi, Tokyo No. Teijin Limited Tokyo Research Center (56) Reference WO 89/6239 (WO, A) PROTEIN ENGINEERI NG, VOL. 3, NO. 3, p. 215 −220 (1990) (58) Fields investigated (Int.Cl. ⁶ , DB name) C12N 15 / 15,15 / 62 C07K 14 / 81,19 / 00 C12N 1/21 C12P 21 / 02,21 / 06 A61K 38/57 CA (STN) REGIDRY (STN)

Claims (27)

(57) [Claims] 1. A elastase inhibitory activity polypeptide de having a portion thereof or their biologically equivalent amino acid sequence having the amino acid sequence or elastase inhibitory activity represented by SEQ ID NO: 1. Wherein elastase inhibitory activity polypeptide de of claim 1 wherein the Cys residue in the amino acid sequence forms a disulfide bond required mutually inhibitory activity. 3. The amino acid sequence according to claim 3, wherein the disulfide bond is Cys ⁶⁴ and Cys ⁹³ , Cys ⁷¹ and Cys ⁹⁷ , Cys in the amino acid sequence.
⁹² and Cys ^80, and Cys ⁸⁶ and elastase inhibitory activity polypeptide de of claim 2, wherein the Cys residues cross-coupling of Cys ^101. Wherein elastase inhibitory activity polypeptides de according to any one of claims 1 to 3 is obtained by genetic recombination techniques. 5. The elastase inhibitory activity polypeptide de having a portion thereof or a biologically equivalent amino acid sequence having the amino acid sequence or elastase inhibitory activity represented by SEQ ID NO: 2. 6. elastase inhibitory activity polypeptide de of claim 5, wherein the Cys residue in the amino acid sequence forms a disulfide bond required mutually inhibitory activity. 7. The disulfide bond forms Cys ⁶⁴ and Cys ⁹³ , Cys ⁷¹ and Cys ⁹⁷ , Cys in the amino acid sequence.
⁹² and Cys ^80, and Cys ⁸⁶ and elastase inhibitory activity polypeptide de of claim 6 wherein the Cys residues cross-coupling of Cys ^101. 8. The elastase-inhibiting polypeptide according to any one of claims 5 to 7, which is obtained by a genetic recombination technique. 9. A compound represented by the following formula [I] AXB... [I] wherein A is human growth hormone or a part thereof,
Is derived from T7 phage gene10 protein or a part thereof.
Represents a carrier protein that; B represents elastase inhibitory activity polypeptide de or Ranaru desired protein of the claim 1 or claim 5, wherein; X is under conditions that do not denature the said purpose protein, chemical or biological 1 represents a crosslinked polypeptide having an amino acid sequence that can be cleaved by a method. ). 10. The fusion protein according to claim 9, wherein the carrier protein is human growth hormone or a part thereof. 11. A carrier protein comprising a Phe at amino acid residue No. 1 to a Phe at position 139 of natural human growth hormone.
10. The fusion protein of claim 9, which is a polypeptide fragment up to: 12. A carrier protein comprising a natural human growth hormone, from Phe at amino acid residue number 1 to Gln at amino acid position 122.
10. The fusion protein of 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 part thereof. 14. X is (Asn-Gly) _n , (Val
-Pro-Arg) _n , (Leu-Val-Pro-A)
r g) _n , and (Asp-Asp-Asp-Asp-L)
ys) _n is a cross-linked peptide or polypeptide having an amino acid sequence selected from the group consisting of: n is an integer of 1 to 10, and Arg at the amino terminus in B is X
The fusion protein according to claim 9, wherein the fusion protein is bound to any of Gly, Arg, Arg, or Lys. 15. A method for producing elastase inhibitory activity polypeptide de of claim 1 or claim 5, wherein the fusion protein of claim 9 represented by the above formula [I], chemical or biological cleavable amino acid distribution Ri by the way
A method comprising cutting column X. 16. The method of claim 15, wherein said chemical method is with hydroxylamine or a homolog thereof. 17. The method of claim 15, wherein said biological method is with thrombin, enterokinase, or a homolog thereof. 18. A human growth hormone or a part thereof,
Or T7 phage gene10 protein or a part thereof
A gene encoding Ranaru carrier protein, portions thereof or elastase inhibitory activity polypeptide having their biologically equivalent amino acid sequence having the amino acid sequence or elastase inhibitory activity represented by SEQ ID NO: 1 or SEQ ID NO: 2 fusion protein gene in which the gene codes, linked via a gene coding for crosslinking peptides with a cross-sectional possible amino acid sequence by chemical or biological methods under conditions which do not denature the desired protein. 19. An amino acid sequence that can be cleaved by a chemical or biological method under conditions that do not denature the target protein, is (Asn-Gly) _n , (Val-Pro-Ar)
g) a bridge having an amino acid sequence selected from the group consisting of _n , (Leu-Val-Pro-Arg) _n , and (Asp-Asp-Asp-Asp-Lys) _n (n represents an integer of 1 to 10) 19. The fusion protein gene according to claim 18, which is a peptide or a polypeptide. 20. A carrier protein comprising the amino acid residue No. 1 Phe to the 139th Phe of natural human growth hormone.
19. The fusion protein gene according to claim 18, which is a polypeptide fragment up to: 21. A fusion protein gene expression type plasmid containing the fusion protein gene according to any one of claims 18 to 20 . A recombinant microbial cell comprising the fusion protein gene according to any one of claims 18 to 20 . 23. A method for producing elastase inhibitory activity polypeptide de of claim 1 or claim 5, the method comprising the Turkey to enzymatic degradation of human leukocyte elastase inhibitor蛋white. 24. An error according to claim 1 or claim 5.
A method for producing a stase inhibitory activity polypeptide, comprising:
Amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2
Is chemically synthesized using the amino acids that make up
How to be. 25. A therapeutically effective amount of elastase inhibitory activity polypeptide de of claim 1 or claim 5, comprising a pharmaceutically acceptable carrier, a therapeutic agent for a disease caused by neutrophil overactivation. 26. The therapeutic agent according to claim 25, wherein the disease is a disease caused by neutrophil-releasing protease. 27. The therapeutic agent according to claim 26 , wherein the disease is an inflammatory disease, platelet aggregation thrombosis, or reperfusion injury after ischemia.