JPH07126294A - New cysteine protease inhibitor - Google Patents

Abstract

PURPOSE:To provide the inhibitor having a high cysteine protease inhibitory effect, capable of inhibiting absorption of bone by osteoclast and useful as a raw material for medicines and foods. CONSTITUTION:A cysteine protease inhibitor has following properties: (1) an affinity to an immobilized carboxymethylated papain. (2) an inhibitory effect on papain. (3) the molecular weight of 16+ or -2KDa (by SDS-PAGE) or 13+ or -2KDa. and (4) the N-terminal amino acid sequence of the sequence no. 1 or 2 in the sequence table.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel cysteine protease inhibitor. The novel cysteine protease inhibitor provided by the present invention has an inhibitory effect on the bone resorption action of osteoclasts and is useful as a raw material for foods and pharmaceuticals for the purpose of suppressing bone aging.

[0002]

2. Description of the Related Art Cysteine protease inhibitors are substances that inhibit the proteolytic activity of cysteine proteases having an SH group at the active center and have been found in animal tissues, cells, blood and urine. Such cysteine protease inhibitors and proteinaceous substances are collectively called cystatin. Cystatin, a cysteine protease inhibitor found so far, is classified into three families based on its molecular structure (Biochem. J., 236, 312 (1986)). According to this document, cystatin β (Biochem. Biophys. Res.
ommun., 115, 902 (1983)), cystatin α derived from rat epidermis (Biochem. Biophys. Res. Commun., 121, 149 (19
84)) and Stefin A (Hoppe-S) found in human leukocytes.
eyler's Z. Physiol. Chem., 364, 1487 (1983)). The molecular weight of each of these cystatins is approximately 12K.
It shows Da and has no sugar.

Family 3 includes cystatin C (Proc. Natl. Acad. Sci. USA 79 , 3024 derived from human urine).
(1982)), egg white cystatin (Hoppe-Seyler's Z. Physiol.
Chem., 364, 1487 (1983)) is classified into this family, and bovine colostrum-derived cystatin (FEBS Lett., 186, 41
(1985)) also belongs to this family. Those belonging to this family have a molecular weight of about 13 to 15 KDa and, like family 1, do not have sugars.

Furthermore, human kininogen, a plasma protein (Biochemistry, 23 , 5691 (1984)), rat T
Kininogen (Biochem. Biophys. Res. Commun., 129,
Kininogens, such as 280 (1985), make up family 3. It is known that kininogens include a high molecular weight kininogen having a molecular weight of 78 KDa and a low molecular weight kininogen having a molecular weight of 45 KDa. The present inventors have isolated a novel cysteine protease inhibitor having a sugar chain and a molecular weight of about 57 KDa from bovine colostrum, and filed a patent application as Japanese Patent Application No. 5-84191.

As one of the useful actions of cysteine protease inhibitors, a virus growth inhibitory action has been confirmed (Biochem. Biophys. Res. Commun., 127, 1
072 (1985)). In addition, since it exhibits an action of suppressing the release of calcium from the bones of animal models of bone diseases, it is disclosed in Japanese Patent Application Laid-Open No. HEI-2.
No. 2,235,29 discloses the use as an anti-allergic agent and a therapeutic agent for bone diseases. In addition, JP-A-61-2
No. 25130 discloses that cystatin derived from egg white is purified,
A technique of using this as an antiviral agent is disclosed.

Further, Japanese Patent Application Laid-Open No. 64-2582 discloses a synthetic gene for cystatin α, Japanese Patent Application Laid-Open No. 1-71492 discloses a synthetic gene for cystatin B, and Japanese Patent Application Laid-Open No. 157390/1991.
Each of the publications discloses a synthetic gene for cystatin A, and it has become possible to produce cystatin by genetic engineering.

In recent years, with the progress of aging, osteoporosis caused by bone resorption of osteoclasts is rapidly increasing. At present, there is a calcitonin preparation as a drug that suppresses the osteoclast resorption activity. However, this preparation is a hormone preparation using a hormone derived from salmon or eel as a medicine, and at present, a safe substance that can be obtained as a food material and can be used as a food material has not been studied so far.

[0008]

[Problems to be Solved] The present inventors have studied cysteine protease inhibitors known to exist in milk so that they can be used as food materials safely and obtained from food materials. We have discovered a new cystatin-like substance that is clearly different from what is known as milk-derived cystatin. This substance has a relatively large molecular weight as compared with cystatin C belonging to the cystatin family 2, and also has a novel N-terminal amino acid sequence not found in the cystatins known so far. Therefore, an object of the present invention is to provide a novel cystatin-like cysteine protease inhibitor derived from milk.

[0009]

The novel milk-derived cystatin-like cysteine protease inhibitors provided by the present invention are identified by the following properties. (1) It has a binding property to the immobilized carboxymethylated papain. (2) It has an inhibitory activity against papain. (3) 16 ± 2K as measured by SDS-PAGE
Da or 13 ± 2 KDa is shown. (4) The N-terminal amino acid sequence represents either the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the sequence listing (The amino acid sequence of SEQ ID NO: 2 of the sequence listing is the N-terminal side of the amino acid residues of SEQ ID NO: 1). The Arg-Pro residue corresponding to the two residues was deleted, starting from the third Gly).

The N-terminal amino acid sequence of the substance of the present invention is obviously different from that of the substance known as cystatin C derived from milk and other substances of the cystatin family. Also, there is no literature reporting such an N-terminal amino acid sequence. The substance of the present invention is a novel protein. The molecular weight of the substance of the present invention is
As described above, 16 ± 2 KDa or 13 ± 2 under both SDS-PAGE, reducing and non-reducing conditions.
It is either KDa. Of these, the substance showing 16 KDa was positive for path staining, and therefore the presence of sugar chains was estimated,
On the other hand, the substance showing 13 KDa does not have a sugar chain because it is negative for Pass staining. From the analysis shown below, it is considered that the substance exhibiting 16 KDa has a sugar chain added to the substance exhibiting 13 KDa.

The substance of the present invention is separated into two fractions having different molecular weights by affinity chromatography with immobilized carboxymethylated papain and gel filtration. By subjecting the separated fractions to reverse phase chromatography, they can be collected as single peaks. When this reverse phase chromatography single peak is subjected to SDS-PAGE, it is still detected as a single band. The molecular weight of this band is the value shown above. A single peak of this reverse phase chromatography was collected, and the N-terminal amino acid sequence was analyzed by an amino acid sequencer (120A type analyzer manufactured by ABI) by the gas phase method. As a result, two amino acid sequences were detected from one peak. This N-terminal amino acid sequence is described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. The coexistence of such sequences is considered to be derived from protein processing in the process of secretion, and the Arg terminus and Gly
It was confirmed that the two types of terminals coexist. This amino acid sequence is common to the substance of 16 KDa and the substance of 13 KDa, and the substance of 13 KDa is considered to exhibit such a molecular weight because it is negative for path staining and no sugar chain is bound.

When compared with kininogen, which is known as a substance having a sugar chain in the cystatin family, the substance having a sugar chain of the substance of the present invention has a molecular weight of 16
± 2 KDa. On the other hand, kininogen has a molecular weight of 78 KDa and high molecular weight kininogen of 45 KDa.
Although a low molecular weight kininogen of Da is known, the substance having a sugar chain of the present invention has a molecular weight of 16 ± 2 KDa, and can be clearly distinguished from kininogen by measuring the molecular weight. Thus, the substance provided by the present invention can be said to be a novel cystatin-like cysteine protease inhibitor. Further, as will be described later, the substance of the present invention is compared with substances of the cystatin family 2 separated from milk,
The inhibitory activities of cysteine proteases show similar values.

The substance of the present invention can be recovered from the milk of mammals such as humans and cows, and particularly easily from bovine milk. The substance of the present invention is contained in a large amount especially in colostrum during the lactation period, but is also contained in normal milk. As described in the above-mentioned literature, these milks can be separated by an operation such as affinity chromatography using affinity with papain, which is a typical cysteine protease.

That is, NaCl was added to bovine defatted colostrum to a final concentration of 0.5 M, and this was subjected to affinity chromatography using a carboxymethylated papain-immobilized carrier to obtain a concentrated fraction of bovine colostrum cystatins. Prepare the minutes. This fraction contains the known cystatin and the substance of the present invention. In particular, the addition of NaCl suppresses non-specific adsorption and makes it possible to prepare a concentrated fraction with high specific activity. Alternatively,
Prepare a whey fraction with an acid, collect the fractions obtained by salting out the acidic whey fraction by ammonium sulfate precipitation, and further desalinate by membrane treatment as the starting material, and use the carboxymethylated papain-immobilized carrier for affinity. Subject to chromatography to prepare a concentrated fraction of bovine colostrum cystatins. Further, this fraction can be separated by chromatography such as gel filtration chromatography to separate the cystatin-like cysteine protease inhibitor which is the substance of the present invention from the known cystatin. In addition to this, other methods such as reverse phase chromatography and HPLC known as protein purification methods may be combined for purification.

The inhibitory activity of the substance of the present invention against papain is
Barrett et al. ("Methods in enzymology" Vol.80 1
981, pp771). That is, 0.
20 mM phosphoric acid containing 1 mM benzoyl-D, L-arginine-4-nitroanilide (Benzoil-D, L-arginine-4-nitroanilide) in dimethylsulfoxide as a substrate and 2 mM EDTA as a buffer for measurement This is a method of determining the enzyme inhibition rate by measuring the enzyme activity by a colorimetric method using a solution in which cysteine was added to a buffer solution so that the final concentration was 8 mM immediately before use. When the inhibitory activity of the novel cysteine protease inhibitor of the present invention is measured using cystatin in milk as a control, the inhibitory activity per protein shows almost the same inhibition rate.

The substance of the present invention is subjected to gel isoelectric focusing (Fast
When the isoelectric point was measured by System Pharmacia), a substance with a pI of 16 KDa showed 4.3-5.2, and a substance with a 13 KDa showed pI of 5.2-5.8.

Of the substances of the present invention, a substance exhibiting 13 KDa and a substance exhibiting 16 KDa are reduced and pyridylethylated, respectively, and hydrolyzed by a conventional method using a protease of Achromobacter litchis or cyanogen bromide to obtain a peptide. When mapping was carried out, all were identical except for the peptide presumed to have a sugar chain attached. From this, it is presumed that the cysteine protease inhibitor of the present invention has a substance having the same amino acid sequence and different N-terminals, and a substance to which a sugar chain is bound and a substance to which a sugar chain is not bound are present. Was done. The deduced primary structure of the amino acid sequence is shown in FIG. 5 and the result of analysis of the amino acid sequence of 13 KDa in which the N-terminal has not been deleted is shown in SEQ ID NO: 5 of the Sequence Listing. The amino acid name of Xaa has not been determined. The result of comparing the homology of this sequence with the known amino acid sequence of the cystatin family is shown in FIG. From these results, the novel cysteine protease inhibitor of the present invention shows a high homology with the consensus region of cystatin family 2, but the amino acid sequences of other sites are different, and it is a novel cysteine protease inhibitor belonging to the cystatin family. It can be said that there is.

The substance of the present invention is a protein resident in milk and is considered to be highly safe. Therefore, it can be used as an antiviral agent, an antiallergic agent, or a bone disease therapeutic agent found in known cystatins, and can be formulated as needed or used alone. It can also be used by mixing it in foods and beverages. When used as a medicine, injections, oral preparations, suppositories, etc. can be exemplified. In addition, protein preparations are often used as injections in general, and they contain a pharmaceutically effective amount of the substance of the present invention, and are pharmaceutically acceptable stabilizers, excipients, isotonic agents, and soothing agents. , A preservative, a buffer and the like can be contained in the preparation.

The present invention will be described in more detail with reference to the following examples.

Example 1 Purification of Novel Cysteine Protease Inhibitor (1) Measurement of Inhibitor Activity
The inhibitory activity against papain was determined by the method of Barrett et al. ("Meth
ods in enzymology "Vol.80 1981, pp771) 0.1 M benzoyl-D, L-arginine-4
-Nitroanilide (Benzoil-D, L-arginine-4-nitroanili
de) Dimethyl sulfoxide solution was used as a substrate, and 20 mM phosphate buffer containing 2 mM EDTA was added as a measurement buffer to a final concentration of 8 mM cysteine immediately before use. Papain at a concentration of 0.5 mg / ml,
It was dissolved in a measurement buffer containing no cysteine. Also,
A 30% acetic acid solution was used as a reaction stopping solution. The measurement was performed according to the following procedure. A phosphate buffer containing 1 ml of cysteine and 0.1 ml of papain solution were placed in a measuring tube and incubated at 37 ° C. for 5 minutes. Then
Sample solution 0.1 ml Substrate solution 30 μl was added and stirred.
After reacting at 37 ° C. for 30 minutes, 0.2 ml of a reaction stop solution was added and the absorbance at 410 nm was measured. The inhibitor activity was calculated by the following formula (I).

Specific activity [unit / mg] = {(A ₀ -A _I ) × 1.43} / {8.8 × 30 × W _S } (I) A ₀ : Inhibitor-free absorbance A _I : Sample absorbance W _S : Amount of protein in sample solution [mg]

(2) Ammonium sulfate fractionation Skim milk was prepared from 9 l of bovine colostrum milked within 1 day after delivery by centrifugation, and acid whey was prepared according to a conventional method.
Ammonium sulfate was added to the whey so that the saturation was 35%, and the resulting precipitate was removed by centrifugation. Ammonium sulfate was further added so that the degree of saturation was 55%, and the resulting precipitate was collected by centrifugation. In order to remove ammonium sulfate contained in the recovered precipitate, DF treatment was performed using a follow fiber type UF membrane (Asahi Kasei) having a cut-off molecular weight of 6 KDa. That is, 5
0 mM phosphate-0.5 M NaCl buffer (pH 7.
It was dissolved in 0) and treated with DF in the same buffer.

(3) Carboxymethylated papain affinity chromatography The carboxymethylated papain was treated with tresyl toyopearl (Tr
esyl-Toyopearl, manufactured by Tosoh)
After being packed in a column of × 150 mm, 50 mM phosphoric acid-0.
Equilibrated with 5M NaCl buffer (pH 7.0). The precipitate fraction with a saturation degree of ammonium sulfate of 35 to 55% was passed through this column to adsorb the cysteine protease inhibitor. After passing, 0.1% Tween was added to the buffer used for equilibration.
The column was washed with a solution containing -20 added. Then 20
The cysteine protease inhibitor was eluted with mM acetic acid-0.5M NaCl solution, and the eluted fraction was immediately adjusted to 1M.
Neutralized with NaOH solution.

(4) Gel Filtration The fraction prepared by affinity chromatography was subjected to gel filtration using Sephacryl S-200HR (Pharmacia). The activity of each fraction is shown in Example 1.
As shown in (1), the absorbance of A ₀ : inhibitor-free and the absorbance of A _I : sample were determined, and the relative activity value was determined by the following formula (II).

Activity [%] = {(A ₀ −A ₁ ) / A ₀ } × 100 (II)

The results are shown in FIG. The fraction prepared by affinity chromatography was concentrated with a follow fiber type UF membrane (Asahi Kasei) having a molecular weight cutoff of 6 KDa. The solution was passed through a column (50φ × 750 mm) equilibrated with 0.1 M ammonium hydrogen carbonate buffer. The eluate was collected, and among the fractions showing inhibitory activity, two peaks (CPI ₁₆ and CPI ₁₃ ) indicated by arrows in FIG. It was subjected to reverse phase chromatography.

(5) Reversed phase chromatography VYDAC 214TP54 (manufactured by Vydac) column (4.6 mm × 25)
Reversed phase chromatography was performed using
It was To equilibrate the column, use 0.08% TFA (trif
5% CH with addition of (loroacetic acid) 3CN-95% H 2 O
80% C with 0.06% TFA added as eluent
H 3CN-20% H Using 2O, 1% / min linear graph
Elute with a gradient. Collect each peak and
Results of activity measurement (Fig. 2), P in the figure₄₀And P₄₁When
Activity was observed in the indicated peaks. Part of the active fraction
As a result of being subjected to reverse phase chromatography under one condition,
Showed a single peak. Lyophilize the two active fractions
Then P₄₀And P₄₁From 0.47 mg, 0.
68 mg of lyophilized preparation was obtained.

[0027]

Example 2 In this example, P ₄₀ obtained in Example 1 was used.
And shows an example of the characteristic values were determined for the two cysteine protease inhibitor of P _41. (1) SDS-PAGE FIG. 3 shows the results of SDS-PAGE of two inhibitors purified by reverse phase chromatography. Both P ₄₀ and P ₄₁ showed a single band, and no impurities were observed. In addition, there was no change under reducing or non-reducing conditions, indicating that both consist of a single polypeptide. The molecular weights of P ₄₀ and P ₄₁ are each about 16
It was confirmed to be KDa, about 13 KDa.

(2) Inhibitor activity The inhibitory activities of purified 16 KDa, 13 KDa and known colostrum cystatin against papain were compared according to the method described in Example 1. The results of the comparative measurement are shown in Table 1.

[0029]

[Table 1] Activity of colostrum cysteine protease inhibitor ────────────────────────── Specific activity CPI ₁₆ ¹⁾ CPI ₁₃ ²⁾ coCys ³⁾ ────────────────────────── [unit / mg] 0.310 0.284 0.270 ───────────────── ────────── 1) 16kDa Cysteine Protease Inhibitor 2) 13kDa Cysteine Protease Inhibitor 3) Colostrum Cystatin

From Table 1, the two newly obtained inhibitors showed the same specific activity as that of the known colostrum cystatin.

(3) N-Terminal Amino Acid Sequence N-terminal amino acid sequence analysis of purified cysteine protease inhibitors having a molecular weight of 16 KDa and 13 KDa was carried out using an amino acid sequencer by a gas phase method. Both of them showed two signals in most cycles, and the signals of both were completely in agreement, which confirmed that both fractions contained the same two-component peptide chains. Further, assuming that two residues of the N-terminus of one of the two components are lacking in the two components, two kinds of sequences having 18 identical residues were obtained. From this, the two fractions used in the analysis each contained two inhibitors, and one of the two inhibitors was
It could be determined that the form lacked the terminal two residues.
This sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. For comparison, the N-terminal amino acid sequence of known colostrum cystatin is shown in SEQ ID NO: 3 in the sequence listing, and the sequence of human cystatin C is shown in SEQ ID NO: 4 in the sequence listing. These known cystatins and the novel cysteine protease inhibitor of the present invention have completely different sequences. The comparative sequences of these four substances are shown in FIG. The N-terminal amino acid sequence of the cysteine protease inhibitor purified by this method showed no homology with known colostrum cystatin or other known cystatins.

(4) Amino acid sequence analysis and primary structure analysis of purified 13 KDa The entire amino acid sequence of the purified 13 KDa cysteine protease inhibitor was analyzed. Reductive pyridylethylation of purified cysteine protease inhibitor,
Achromobacter lyticu
s) Protease (API) or cyanogen bromide (CNB)
Hydrolysis with r), followed by separation of the peptides by reverse phase chromatography for amino acid sequence analysis. Amino acid sequence analysis was performed as in (3) above, and all 121
The amino acid sequence was confirmed. In the portion corresponding to the N-terminal amino acid, two peaks are observed as described in (3),
One started with glycine and one was unable to determine the N-terminal amino acid. The sequence of 121 amino acids is shown in FIG. 5 and SEQ ID NO: 5 in the sequence listing.

This sequence was compared with known cysteine protease inhibitors belonging to the cystatin family. The respective amino acid sequences are shown in FIG. In the sequence, there were two phosphorylation sites (# 14 Ser, # 35 Ser) by casein kinase (casein kinase II), and it was presumed to be a phosphorylated protein. It also contained a consensus sequence for the cystatin family. 13KD
a Cysteine protease and cystatin family 2
The homology of SWISS-PORT (R24.0 D
The analysis was carried out by using ember 1992), and the results shown in Table 2 below were obtained. It was confirmed that the protein is different from the known cystatin family.

[0034]

[Table 2] Homology of known family 2 cystatin to cysteine protease inhibitor of the present invention ───────────────────────────────── Cysteine protease Inhibitor% ──────────────────────────────── Egg white cystatin 36.5 Colostrum cystatin 34.5 Human cystatin C 33. 9 Rat cystatin C 42.4 Human cystatin S 33.0 Human cystatin SN 38.0 Human cystatin SA 31.2 Rat cystatin S 32.8 Human cystatin D 31.0 ─────────────── ────────────────────

(4) Other characteristics The presence or absence of sugar chains of the purified 16 KDa and 13 KDa cysteine protease inhibitors was confirmed by PAS staining. As a result, it was confirmed that only the 16 KDa inhibitor was positive and contained a sugar chain.

Both images were subjected to gel isoelectric focusing (Fast
system Pharmacia) to determine the pI value. As a result, the pI value of the 16 KDa inhibitor was 4.3-
5.2, the pI value of the 13 KDa inhibitor is:
It was 5.2-5.8. From the above results, it was revealed that the substance of the present invention is a substance having two molecular weights of a sugar chain-containing molecular weight of 16 KDa and a sugar chain-free 13 KDa, and has two types of N-terminal amino acid sequences.

[0037]

INDUSTRIAL APPLICABILITY The present invention provides a novel cysteine protease inhibitor. The substance provided by the present invention is a novel substance having a different N-terminal amino acid sequence and total amino acid sequence compared to known cystatin, has an inhibitory effect on the bone resorption action of osteoclasts, and suppresses bone aging. It is useful as a food material or a drug material for the purpose.

[0038]

[Sequence Listing] SEQ ID NO: 1 Sequence length 22 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Pro Gly Asp Arg Lys Val Gly Glu Leu Gln Glu Leu Ser Pro Asn 1 5 10 15 Asp Pro Gln Val Gln Lys 20

SEQ ID NO: 2 Sequence length 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Asp Arg Lys Val Gly Glu Leu Gln Glu Leu Ser Pro Asn Asp Pro 1 5 10 15 Gln Val Gln Lys 20

SEQ ID NO: 3 Sequence length 22 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Leu Leu Gly Gly Leu Met Glu Ala Asp Val Asp Glu Glu Gly Val 1 5 10 15 Gln Glu Ala Leu Ser Phe 20

SEQ ID NO: 4 Sequence length 22 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu Glu Gly Val 1 5 10 15 Arg Arg Ala Leu Asp Phe 20

SEQ ID NO: 5 Sequence length 121 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Xaa Pro Gly Asp Arg Lys Val Gly Glu Leu Gln Glu Leu Ser Pro Asn 5 10 15 Asp Pro Gln Val Gln Lys Ala Ala Gln Val Ala Val Ala Asn Tyr Asn 20 25 30 Met Gly Ser Asn Ser Asp Tyr Tyr Tyr Arg Asp Ile Thr Ile Leu Arg 35 40 45 Ala His Ser Gln Leu Val Ala Gly Ile Lys Tyr Tyr Leu Thr Val Asp 50 55 60 Met Glu Ser Thr Ala Cys Arg Lys Ser Ala Val Ala Gly Asp His Val 65 70 75 80 Asp Leu Thr Thr Cys Pro Leu Ala Ala Glu Ala Gln Gln Glu Lys Leu 85 90 95 Arg Cys Asp Phe Glu Ile Leu Val Val Pro Trp Lys Asn Ser Ser Gln 100 105 110 Leu Leu Lys Trp Asp Cys Val Ser Leu 115 120

SEQ ID NO: 6 Sequence length 119 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Asp Arg Lys Val Gly Glu Leu Gln Glu Leu Ser Pro Asn Asp Pro 5 10 15 Gln Val Gln Lys Ala Ala Gln Val Ala Val Ala Asn Tyr Asn Met Gly 20 25 30 Ser Asn Ser Asp Tyr Tyr Tyr Arg Asp Ile Thr Ile Leu Arg Ala His 35 40 45 Ser Gln Leu Val Ala Gly Ile Lys Tyr Tyr Leu Thr Val Asp Met Glu 50 55 60 Ser Thr Ala Cys Arg Lys Ser Ala Val Ala Gly Asp His Val Asp Leu 65 70 75 80 Thr Thr Cys Pro Leu Ala Ala Glu Ala Gln Gln Glu Lys Leu Arg Cys 85 90 95 Asp Phe Glu Ile Leu Val Val Pro Trp Lys Asn Ser Ser Gln Leu Leu 100 105 110 Lys Trp Asp Cys Val Ser Leu 115

[Brief description of drawings]

FIG. 1 shows a fractionation pattern of cysteine protease inhibitor by gel filtration.

[Explanation of symbols]

 ─ ● ─ Indicates activity. ─── Indicates the absorbance at OD280 nm.

Figure 2: Reversed phase HPLC of fractions obtained by gel filtration.
The pattern is shown. This is a simultaneous description of the patterns obtained by subjecting each of the fractions shown in FIG. 1 to reverse phase HPLC.

FIG. 3 shows the SDS-PAGE pattern of the cysteine protease inhibitor of the present invention fractionated by HPLC.

[Explanation of symbols]

1 pattern of reducing state of 16 KDa cysteine protease inhibitor 2 pattern of reducing state of 13 KDa cysteine protease inhibitor 3 pattern of non-reducing state of 16 KDa cysteine protease inhibitor 4 shows pattern of non-reducing state of 13 KDa cysteine protease inhibitor

FIG. 4 shows a comparison of the N-terminal amino acid sequences of the cysteine protease inhibitor of the present invention, the known substances colostrum cystatin and human cystatin C.

[Explanation of symbols]

a: N of cysteine protease inhibitor of the present invention
Terminal amino acid sequence b: N of cysteine protease inhibitor of the present invention
Terminal amino acid sequence c: Colostrum cystatin N-terminal amino acid sequence d: Human cystatin C

FIG. 5 shows the primary structure of the amino acid sequence of a 13 KDa cysteine protease inhibitor according to the present invention.

FIG. 6 shows a homology diagram of the 13 KDa cysteine protease inhibitors according to the invention and the amino acid sequence of the known cystatin family up to the 40th amino acid.

FIG. 7 shows a homology diagram of the 13 KDa cysteine protease inhibitor according to the present invention with the amino acid sequences 41-85 of the known cystatin family.

FIG. 8 shows a homology diagram of the 13 KDa cysteine protease inhibitor according to the present invention with the amino acid sequence 86 to 121 of the known cystatin family.

Claims (4)

[Claims] 1. A cysteine protease inhibitor having the following properties. (1) It has a binding property to the immobilized carboxymethylated papain. (2) It has an inhibitory activity against papain. (3) 16 ± 2K as measured by SDS-PAGE
Da or 13 ± 2 KDa is shown. (4) Sequence listing It has the N-terminal amino acid sequence represented by SEQ ID NO: 1. 2. A cysteine protease inhibitor having the following properties. (1) It has a binding property to the immobilized carboxymethylated papain. (2) It has an inhibitory activity against papain. (3) 16 ± 2K as measured by SDS-PAGE
Da or 13 ± 2 KDa is shown. (4) Sequence listing It has the N-terminal amino acid sequence represented by SEQ ID NO: 2. 3. A cysteine protease inhibitor having the following properties. (1) It has a binding property to the immobilized carboxymethylated papain. (2) It has an inhibitory activity against papain. (3) 16 ± 2K as measured by SDS-PAGE
Da or 13 ± 2 KDa is shown. (4) Sequence Listing Contains the amino acid sequence of SEQ ID NO: 5. 4. A cysteine protease inhibitor having the following properties. (1) It has a binding property to the immobilized carboxymethylated papain. (2) It has an inhibitory activity against papain. (3) 16 ± 2K as measured by SDS-PAGE
Da or 13 ± 2 KDa is shown. (4) Sequence Listing Contains the amino acid sequence of SEQ ID NO: 6.