JPH03219871A - Serine protease - Google Patents

Abstract

PURPOSE:To obtain serine protease having an activating action on kalliklein- quinine system in human blood plasma, capable of giving a vaccine or an antibody, etc., effective for allergic disease owing to Dermatophagoides farinae in a low cost and having specific physicochemical properties and molecular weight. CONSTITUTION:The subject serine protease exists in a culturing medium of Dermatophagoides farinae and has analyzed values of amino acid obtained by a hydrochloric acid hydrolysis and expressed in the table I, and has amino acid sequences of from chain terminal to the 11th element and from the 13th element to the 20th element, respectively expressed by formula I and formula II. Said serine protease is able to be clearly distinguished from formerly separated substance from Dermatophagoides farinae in the viewpoints of amino acid composition and amino acid sequence. Whereas, said serine protease exhibits same migration pattern by SDS-PAGE in the presence of absence of beta- mercaptoethanol as a reducer and migrates as a single band by staining with silver, thus is identified as a single protein.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to Dermatophagoides f., which is considered to be one of the causes of allergies.
The present invention relates to a novel serine protease capable of activating the kallikrein-kinin system in normal human plasma obtained from a medium extract of M. arinae' and a method for purifying the same.

[Conventional technology]

Arachnididae belongs to the arthropods, Arachniformes, order Dani, family Chirididae, genus Arachnididae,
Dermatophagoides pterony
It is said that together with S. ssius, they account for about 60 to 80% of the mites in indoor dust.

Derl (molecular weight: 25.000) and Der2 (molecular weight: 12.000) are present as major allergens in the Dermatophagoides extract, and are known to exhibit strong antigenicity to [gE antibodies] in patients with mite allergies. [It is known that histamine and SR3 released from mast cells when an allergen binds to gE antibodies cause allergic inflammation.

On the other hand, Rikurenginin system (hereinafter referred to as KK- system) 1
Hayashi [breathing, 7. (12), 1349-1357
．． (1988)], it is said that it is involved in the onset of allergic diseases, and in particular, kinin, which is formed from kininogen by the action of kallikrene, contains bradykinin (BK), which consists of nine amino acids, and bradykinin (BK), which consists of nine amino acids, and one amino acid.
There are two types of kallidins consisting of 0 kinins, and both have similar effects: 1) peripheral vasodilation and associated blood pressure lowering effect 2) contraction of most smooth muscles including bronchial smooth muscle 3) Source of vascular permeability 4) In addition to its inherent properties such as pathogenesis, arachidonic acid, its metabolites, and platelet activating factor (plat
elet activating factor:PA
It amplifies the release and production of histamine caused by F) and increases the release of arachidonic acid and the production of its metabolites and PAF in human platelets and cultured human vascular endothelial cells, and is attracting attention as a mediator of allergic and inflammatory reactions. Kallikrene not only releases kinin from kininogen, but also has activity as a neutrophil chemotactic factor (NCF), and aggregates neutrophils in inflammatory foci. In addition, the plasma KK-system may be involved in various biological regulatory systems such as the endogenous blood coagulation system, fibrinolytic system, complement system, prostaglandin system, and renin-angiotensin system, triggered by the activation of Hageman factor. However, the relationship between this KK system and Dermatophagoids or its extracts has not been made clear, and the substances or enzymes that prove this relationship are completely unknown.

Some proteases derived from mites have been isolated from Dermatophagoides farinae and Dermatophagoides farinae (Stewart G, A, et al.

LANCET, July 15. 154 (1989
)].

[Invention or problem to be solved]

From this point of view, the present inventor has determined that the KK- system is activated by the KK- system (Hagemann factor activation, release of kallikrene from preka IJcrene, M release of kinin from kininogen, etc.)
As a result of intensive research, we aimed to find a substance that can alleviate the inflammation, elucidate the mechanism of action of allergic inflammation involving the KN- system, and provide a method to search for a substance that can alleviate this inflammation. We confirmed the presence of serine protease that activates the KK- system in the culture medium of Dermatophagoides farinae, and established a method to easily isolate it from the culture medium of Dermatophagoides in an unpurified aqueous solution using affinity chromatography. The present invention has now been completed.

[Invention or problem to be solved]

The present invention relates to a serine protease present in the rearing medium of Dermatophagoides nigricans, which has the following physical and chemical properties, has a molecular weight of 30.000, and has a system-activating effect on KK- in human plasma.

■ Amino acid composition analysis value by hydrochloric acid hydrolysis method or serine protease listed in Table 1 below.

Table 1 Asx 10.7 Thr 3.4Ser
9.4 Glx 10.7Gly 12.
0Ala 6.7Val 9.9 1/2Cy
s3. OMet 1.3 11e
6.5 Leu 5.8 Tyr 4.8P
he 0.9Lys 6.0His 1.
3 Arg 3.0Pro 3.2'
Trp 1.3■ Amino acid sequence from the 11th to the NH2 terminal and from the 13th to the 20th or formula (1)
and a serine protease according to formula (2).

Further, the present invention is directed to Dermatophagoides farina (Dermatophagoides farina).
Contains the use of affinity chromatography that specifically acts on the unpurified aqueous solution containing the unpurified aqueous solution extracted from the rearing medium of e) at least in the purification process of the unpurified aqueous solution. The above-mentioned problems can be solved by the method for purifying an unpurified aqueous solution.

Wear.

In the present invention, the representation of amino acids in the serine protease and amino acid composition shall be in accordance with the abbreviations in the amino acid nomenclature adopted by IUPAC or the representation of amino acid residues by abbreviations commonly used in the field.

The physiological activity of the serine protease of the present invention, that is, the activating effect on the KK-system in human plasma, is determined by the method detailed in the Examples below.

The serine protease of the present invention has the amino acid sequences represented by formulas (1) and (2) in the amino acid sequence constituting the enzyme, and the amino acid composition of the enzyme is as listed in Table 1. And the enzyme is KK in human plasma.
- It is specified in that it has an activating effect on the system.

The serine protease of the present invention is clearly differentiated from that previously isolated from Dermatophagoides farinae in terms of its amino acid composition and amino acid sequence [Heymanm P,
W, et a1, J, allergy cl
in.

Immuno1, 83.1055 (1989)]
, and the serine protease of the present invention is 5DS-PA in the presence or absence of the reducing agent β-mercaptoethanol.
By GE, they all showed the same migration pattern and were migrated as a single hand by silver staining, and from this, they were identified as a single protein.

The serine protease of the present invention is considered to be a completely new protease, unlike known enzymes, as it has the following various properties.

l) Action: The serine protease of the present invention can be used in Tris-HCl buffer (p
In addition to having a hydrolytic effect on BOC-Gin-Gly-Arg-MCA, a synthetic substrate for blood coagulation factor XIIa in H7, 4), the method of Morita et al.
Biochem, 82.1495. (1977)
] When measured using a modified method, human plasma KK-
It also has the effect of activating the system.

2) Substrate specificity: The serine protease of the present invention was incubated at 37°C in Tris-HCl buffer (pH 7,4) with the addition of substrates for other enzymes, and when looking at the hydrolysis effect on each substrate, it was found that urokinase, urokinase, It has little effect on substrates such as plasma receptors, and efficiently hydrolyzes substrates of plasma coagulation factors XI[a, Xa, and XIa, particularly substrates of blood coagulation factor XIIa.

3) Optimal pH and Optimum Temperature Looking at the influence of pH at 37°C on the hydrolytic action of the serine protease of the present invention, and the influence of temperature in a Tris-HCl buffer solution of pH 7.4, the pH is 7.
3 to 7.6, especially 7.4, and the temperature is preferably 45 to 53°C, especially 50°C.

4) Inhibitor The influence of various drugs on the hydrolytic action of the serine protease of the present invention was investigated using a Tris-HCl buffer (pH 7, 4).
), substrate B of blood coagulation factor XIIa at 37°C.
Looking at the hydrolysis effect on OC-Gln-Gly-Arg-MCA, phenylmethylsulfonyl fluoride (
PMSF), p-aminobenzamidine, soybean and lima bean trypsin inhibitors, leupepsin, etc., but not EDTA, CaCZ2. Based on these results, the enzyme of the present invention is considered to be a type of serine protease.

Details of these are as shown in Examples below.

The serine protease of the present invention has a unique human plasma KK
Since it activates the KK- system in human plasma, it is useful for elucidating the activating factors of the KK- system in human plasma, and by using the antibody with the serine protease of the present invention as an antigen, it is possible to treat various allergies. It is effective in diagnosis and in suppressing or alleviating allergic symptoms.

The method for producing and purifying the serine protease of the present invention will be described in detail below.

The serine protease of the present invention can be purified, for example, from a rearing medium of the Dermatophagoids nigricans. In this case, in the present invention, the mite breeding medium includes the mites themselves.

The medium, that is, the feed used in the present invention is not particularly limited as long as it is capable of surviving or reproducing Dermatophagoides nigricans; examples thereof include grain flour, fish meal, and dried yeast.

Furthermore, since the mites are saturated in the rearing medium in 3 to 4 months, it is preferable to use a mite rearing medium that has been cultured for the same period of time in order to obtain more of the desired product.

A liquid fraction containing the desired serine protease can be obtained by extracting and separating the mite rearing medium obtained by the above culture according to a conventional method. In particular, when considering the subsequent treatment of the serine protease of the present invention, aqueous solvents such as purified water, weakly alkaline aqueous solutions, weakly alkaline buffers, etc. are used, and in particular, the weakly alkaline buffer is preferably p
Any extract solution having H7 to H9 may be used, such as a Tris-HCl buffer.

Production of a crude sample from the liquid fraction thus obtained can be carried out by appropriately combining various conventionally known operations. Examples of the above operations include treatment using a protein precipitant such as ammonium sulfate salting out, centrifugation, dialysis, ultrafiltration, and concentration.

Further, the purification operation of the crude sample can be performed using various known methods, but it is preferable to use at least affinity chromatography using a carrier that specifically acts on the serine protease in the crude sample. In addition to this, various known means can be combined. Known means include liquid chromatography, electrophoresis, chromatofocusing, etc., particularly gel filtration, ion exchange chromatography, high performance liquid chromatography, reversed phase liquid chromatography, or a combination thereof.

For example, p-aminobenzamidine-Sepharose CL
-Affinity chromatography using 4B as an adsorption carrier, DEAE-) Yopal 650-1 (manufactured by Tosoh Corporation)
Cation exchange chromatography using Sephadex G75 (manufactured by Pharmacia), gel filtration using Sephadex G75 (manufactured by Pharmacia), etc. can be carried out.

In this way, the serine protease of the present invention can be isolated and obtained.

〔Example〕

Hereinafter, specific embodiments of the present invention will be described, or the present invention will be explained below.
It is not limited to this.

1) Hydrolysis activity measurement method for Boc-Gln-Gly-Arg-MCA and measurement method of total protein amount i) Hydrolysis activity side standard method 10 Hydrolysis activity was determined by Youbata et al. (Eur, J., Bioche
17217 (1988)] was modified. Namely, at 37°C, 15μN1 of the synthetic substrate Boc-Gin-Gly for blood coagulation factor XKa
-50m including Arg-MCA (Peptide Institute)
Add about 10 μ! of the sample to 2 ml of M Tris-HCl buffer (pH 7°4), and from the time of stirring Ex380 nm, Em
By measuring the fluorescence intensity at 460 nm over time and determining the release rate of the 7-amino-4-methylcoumarin (AMC) group, the synthetic substrate Boc-Gin-G in the sample was determined.
Hydrolytic activity towards ly-Arg-MCA is obtained. The activity of hydrolyzing 1 μmol of the substrate in 1 minute was defined as IU.

ii) Total protein measurement method Protein quantification in each operation was performed using bovine serum albumin (B
SA) was used as the standard, and the method of Lowry et al. (J, Bio1,
Chem, 193.265 (1951)].

2) Preparation of cultivation medium for Kona hypodermis The rearing of Kona hypodermic mites was carried out by Sasa et al. (Jap, J.

Exp, Med, 40.367, (1970)
] (7) Method. That is, approximately 300,000 subcultured Dermatophagoides mites were inoculated into approximately 330 g of a medium containing 1 part by weight of fish meal and 1 part by weight of dry yeast, and the mixture was incubated at a temperature of 25°C.
A mite rearing medium (number of mites in the medium or approximately 1.5XIO'/g medium) was prepared by culturing for about 4 months at C1 humidity of 65%.

3) Preparation of a crude sample by liquid extraction and concentration After freeze-drying approximately 330 g of the mite breeding medium obtained in 2) above, it was defatted with 1.5% diethyl ether, filtered, and the solid fraction was dried under reduced pressure. Ta. This dry solid fraction was mixed with 50mM)'Jsu-
Add to acid buffer (pH 7°4) 1.31 and incubate at 4°C for 1.
After being immersed for 2 hours, centrifugation was performed at 10,000×g for 20 minutes to obtain a supernatant of approximately 1.01%. Furthermore, 95% of the supernatant
Solid ammonium sulfate was added to achieve saturation, and after standing at 4°C for 24 hours, centrifugation was performed at 10,000×g for 20 minutes to obtain a precipitate. This was dialyzed against the same buffer to obtain a concentrated fraction.

4) Preparation of serine protease The serine protease of the present invention has a system activating effect on KK- in human plasma, and also has a Boc-Gin-Gly-Arg
- Also has MCA hydrolyzing action. Therefore, in order to prepare serine protease, the following operations were carried out, and the above-mentioned hydrolytic activity and total protein amount were measured for each operation using the methods described above.

i) Ammonium sulfate salting out Supernatant obtained in 3) above about 1. Solid ammonium sulfate was added to OI until it was about 60% saturated, the precipitate was separated, and solid ammonium sulfate was further added to it until it was 80% saturated, and it was left at 0°C for 12 hours.
The mixture was centrifuged at 000Xg for 15 minutes to obtain a precipitate. 5m of this
Dissolved in 60 m Tris-HCl buffer (pH 7,4),
Dialysis was performed three times against the same buffer solution 101 to obtain a concentrated solution of 76-.

ii) Affinity chromatography After adding 44 ml of the concentrated culture solution obtained in step i) above to 44 ml of IM NaC 15 mM Tris-HCl buffer (pH 7,4), Holman et al. [J, Bio 1, Chem, 2
51.1663 (1976)]
p-Aminobenzamidine-Sepharose CL-4B with aminocaproic acid as a spacer (φ1.5
m) was added to a column equilibrated with 0.5M Na (J -5mM) Lis-HCl buffer (pH 74) at 4°C, and the non-adsorbed fraction was added to the column equilibrated with 600 m (!) of the same buffer. After washing, no hydrolytic activity was observed in this fraction.Then, the adsorbed fraction was diluted with 0.2M NaCl 5mM Tris-HCl buffer containing Ip-aminobenzamidine (0.15%).
pH 7,4) was eluted at 20m (!), both of which had hydrolytic activity.This eluate was diluted with 5mM)
Dialysis was repeated four times against 1 Offi of hydrochloric acid buffer (pH 7,4) to obtain active fraction 1.

1ii) Cation exchange chromatography DEAE-Toyopearl 6 obtained by equilibrating the active fraction 110mj obtained in ii) above with 5mM Tris-HCl buffer (pH 7,4)
514 (manufactured by Tosoh Corporation).

When the non-adsorbed fraction was washed away with the same buffer solution of 120 m (φ1.5 x 27 cm), no hydrolytic activity was observed in the non-adsorbed fraction. Linear gradient elution was performed from 0.29f NaCl to 5mM) Lis-HCl buffer (pH 7.4), and active fractions eluted around 40-70mM NaCl were collected.

This active fraction was added to 1mM Tris-HCl buffer (pH 7,
4) Repeated dialysis against IOA twice, freeze-dried,
It was dissolved in 2 d of water to obtain active fraction 2.

iv) Gel filtration (Sephadex G-75) above ii
) Triton X-100 was added to 2Lml of the active fraction obtained in
(manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) to a final concentration of 1 (v/v)%, and 50 m M NH41 (Sephadex G- equilibrated with CO3) containing 0.01% Triton X-100. 75 (φ1.5 x 28 cm), fractionated with 0.85 m!!/tube, collected both fraction numbers 30 to 36, and diluted 10 times with phosphate saline (PBS, pH 7,4) IA. The active fraction 3 was obtained by repeating dialysis twice, freeze-drying, and dissolving in 1.1 mM water.

■) Gel filtration (Sephacryl S-200) above iv)
31.1mM of the active fraction obtained was added to Sephacryl S-200 (φ1.5 x 71cm) equilibrated with PBS, fractionated with 1.87711/tube, and fraction numbers 36 to 41 were separated from both fractions. The hydrolytic activity was collected and a single peak was obtained.

The results of gel filtration using Sephacryl S-200 are shown in FIG.

In the figure, the lower horizontal axis is the fractional number, and the vertical axis is the absorbance at 28 cm and the substrate Boc- for blood coagulation factor XIIa.
Gin-Gly-Arg-5 (shows hydrolytic activity in CA).

In addition, as a molecular weight marker, BSA (molecular weight: 1'
v (w65.000), Pseudomonas fragi (P
lipase Q derived from S. fragi (
w:33.000) and liphonuclease A (^(w:
11.000) using the same column is shown on the upper horizontal axis. A summary of each of the above purification steps is shown in Table 2 below.

5) Measurement of hydrolytic activity for each substrate Various synthetic substrates listed in Table 3 were added to 2 J of 5 mM Tris-HCl buffer (pH 7,4) at 37°C to a concentration of 15 μM, and a fixed amount of serine protease was added. (usually 5 to 10 μl) and immediately after stirring,
The fluorescence intensity of the substrate Boc-Gln-Gly-Arg-MC was measured over time to determine the release rate of the AMC group.
The hydrolysis activity of serine protease for each synthetic substrate was determined as a relative rate of hydrolysis for A.

Furthermore, the serine protease of the present invention has a substrate Boc-Gl.
It has a hydrolyzing activity of 158 U/■ protein against n-Gly-Arg-MCA, and Table 3 shows the relative activity when this activity is taken as 100.

Table 3 Synthetic substrate Boc-Gin-Gly-Arg-MCABoc-11
e-Glu-Arg-MCABoc-1al-Leu-
Lys-MCABoc-Glu-Lys-Lys-8 (
CABoc-Giu(OBzl)-Ala-Arg-rufcABoc-Val-Pro-Arg-MCAPy
r-Gly-Arg-MCA Bz-Arg-MCA Z-Phe-Arg-MCA Z-Arg-Arg-MCA Gl t-Gly-Arg-111cASuc-Ala
-Ala-Ala-λIcAArg-MCA Lue-MCA Lys-Ala-MCA Gly-Pro-NICA Pyr-MCA Suc-Leu-Leu-Val-Tyr-MCALy
s-MCA specific activity 100.0 73.8 42.9 38.1 26.2 21.4 13.5 4.8 3.0 2.3 1.2 1.1 0.6 0.3 0.3 0.0 0 0.0 0, O 6) Effect of various substances on hydrolysis activity Various substances listed in Table 4 were added to 5mM Tris-HCl buffer (pH 7 , 4) 2 m
l plus serine protease (usually 5-10 μl)
After addition, the mixture was left at 37°C for 11 hours, and the hydrolysis activity for Boc-Gin-Gly-Arg-MCA substrate (substrate for blood coagulation factor It is shown in Table 4.

The serine protease of the present invention (no drug added) has a hydrolysis activity of 36.7 U/■ protein for the substrate, and Table 4 shows the results when this activity is taken as 100.

Table 4 Substance Name Specific Activity No addition (control) 100 phenylmethylsulfonyl fluoride (PMSF) (1mM)
1, Op-aminobenzamidine (0,9
8m~ri 1.2 (1.9mM) 0.0 Soybean trypsin inhibitor (0.97■/ml) 0
．． 0 Lima bean trypsin inhibitor (0,97■/ml)
0.0 monoiodoacetic acid (1mM)
98.10 Ipepsin (0,97mg/
ml) 0.0 ovomucoid (0,97
■/ml) 0.0 Phosphoramiton (0
, 97mg/ml) 60.0 bovine serum albumin
(1,0■/m1.) 100 ethylenediaminetetraacetic acid (5'mM) 92.2 calcium ion
(10mM) 93.87) 5O3-PA
Analysis by GE electrophoresis Sodium dodecyl sulfate (SD
S)-Polyacrylamide gel (PAGE) is PAGE
Silver staining was carried out using a 15% Silvestin kit (manufactured by Nacalai Tesque). Said 4
5DS- for the serine protease obtained in )■)
The PAGE result is a molecular weight marker. Cytochrome C monomer 0h12.400), the same dimer (Mw2
4.800), the same trimer QIw 37.200), the same tetramer QIW 49.600), and the same hexamer (M
w74.400), the molecular weight of serine protease is about 30.
．． It was recognized as 000 Dalton.

Furthermore, as a result of repeating the serine protease in the presence and absence of the reducing agent β-mercaptoethanol, the electrophoresis pattern was the same and it was electrophoresed as a single band by silver staining. From this fact, the serine protease of the present invention was identified as a single protein without a subunit structure.

8) Measurement of optimal pH and temperature a) Optimum pH Tris-HCl buffer (pH 5-10) at 37°C
Synthetic substrate Boc-Gln-Gly-Arg-MCA
The optimum pH was approximately 7.4 as a result of measuring the hydrolytic activity against. The results are shown in FIG. In the figure, the vertical axis shows the hydrolysis activity as a ratio to the maximum value, and the horizontal axis shows the pH.

b) Tris-HCl buffer (pH 7,
As a result of measuring the hydrolysis activity for the substrate Boc-Gln-Gly-Arg-MCA in 4), the optimum temperature was approximately 50°C. The results are shown in FIG. In the figure, the vertical axis shows the hydrolysis activity as a ratio to the maximum value, and the horizontal axis shows the temperature.

9) Activation of kallikrein-kinin system a) Contraction of rat smooth muscle 0.2 mg of β-nisterradiol (Sigma) was intraperitoneally administered to virgin rats (140-60 g),
Four hours later, the uterus was removed. Below, Kato et al.'s method [! +1
ethOd in Enzymology Vol 8
0. (L., Lorland Ed,).

Detection of bradykinin was carried out according to the method described in 173 (1981)], and the influence of serine protease on the KK- system in human plasma was investigated.

That is, the excised rat uterus was fixed in a circulating constant temperature bath containing 10 mff of Munsick solution, and a biological displacement meter KN-259 and a body recorder KN-
260 (Natsume Seisaku rupture) was used to measure the contraction of isolated smooth muscle caused by bradykinin.

It was confirmed that 5 ng of bradykinin (Peptide Institute) was added to a thermostatic bath and that uterine smooth muscle contracted. After thoroughly washing the uterus with Muncik's solution, 25 μl of normal human plasma (G
eorge KingBio-Medica1, In
c,) and 150 ng of serine protease,
Similar contractions were confirmed. Furthermore, it was confirmed that the interval at which smooth muscle contracts changes depending on the concentration of the protease added. Blood coagulation factor XIIa deficient plasma (G
eorge KingBio-Medica1, I
A similar contraction phenomenon was observed when this protease was added to normal plasma alone, this protease alone, and even prekallikrein-deficient plasma (George).
No smooth muscle contraction was observed when KingBio-Medica 1, Inc.) and serine protease were added.

The results are shown in FIG.

In Figure 4, the vertical axis shows the contraction width of the rat smooth muscle, and the horizontal axis shows time.
It was a minute. A is the result of adding human normal plasma and serine protease, B is prekallikrene deficient plasma and serine protease, and C is the result of adding blood coagulation factor Xna deficient plasma and serine protease.

b) Activity towards kallikrein substrate Z-Phe-Arg, a substrate of human plasma kallikrein
Quantifying kallikrein released from prekallikrein in human plasma by hydrolyzing activity against MCA,
The effect of serine protease on human plasma KK-system was investigated.

i.e. 50 mM) Lis-HCl buffer (pH 7,4) containing 0.1% bovine serum albumin at 37°C.
, 2 mM Z-Phe-Arg-MCA
(Peptide Research Institute) 5μm, human plasma 19μ11 serine protease lμp was added and mixed, and Ex38
Fluorescence intensity at 0 nm and En460 nm was measured over time using a fluorometer. Measurements were similarly made for other samples, and the results are shown in FIG.

In the figure, the horizontal axis shows the reaction time (minutes), and the vertical axis shows the amount of free 7-amino-4-methylcoumarin (nM). Curve A is human plasma and serine protease (1.4 ng), curve B is human plasma and β-blood coagulation factor XI [a, curve is human plasma, and curve E is β-blood coagulation factor XI [a]. a
Factor and curve F are the measurement results when serine protease was added.

10) Amino acid composition of serine protease Transfer approximately 150 μl of the purified serine protease solution to a hard glass sample tube (
Nichiden Rika Glass Co., Ltd. 6 x 40 mm), put it in a reaction vial for hydrolysis (Pierce Co., Ltd.), dried it under reduced pressure, and dried it with 6N-
100 μl of hydrochloric acid (containing 4% thioglycolic acid) was placed in the reaction vial, sealed under reduced pressure, and hydrolyzed at 110° C. for 22 and 72 hours.

After decomposition, the hydrochloric acid was dried under reduced pressure, dissolved in 100 μl of distilled water, transferred to a sample tube for amino acid analysis, and 90 μl of the
The amino acid composition was analyzed by automatically injecting into a Model 35 amino acid analyzer (manufactured by Hitachi). Note that the ninhydrin method was used as the detection method. Tryptophan is not detected with this method. In addition, each separated and identified amino acid was quantified using a calibration curve prepared using standard amino acids (1 nmole), and corrected by taking the average of 22 and 72 hours.

The results obtained are shown in Table 1 above.

11) N-terminal amino acid sequence of serine protease Purified serine protease solution (approximately 50μI!/ml>approximately 2
Using 00 μl, the sequence from the N-terminus to 11 amino acid residues was identified using a gas phase protein sequencer (Model 474, manufactured by Applied Biosystems). Also 1
Sequences from 3 to 20 amino acid residues were also identified at the same time.

The PTH-amino acid solution obtained in each reaction cycle was vacuum centrifuged to dryness, dissolved in a 33% acetonitrile aqueous solution, and separated and identified using HPLC for P'TH-amino acid analysis (Model 12OA, manufactured by Applied Biosystems).

According to the above, the sequence up to 11 amino acid residues from the N-terminus is as shown in the above formula (1), and the sequence from 13 to 20 amino acid residues is as shown in the above formula (2).

〔Effect of the invention〕

The novel serine protease of the present invention can be obtained by a very simple method,
It can be isolated in large quantities and at low cost using a simple device, and vaccines and antibodies effective against allergic inflammation caused by Dermatophagoides can be provided at low cost.

[Brief explanation of drawings]

Figure 1 shows crudely extracted sephacryl 8 containing serine protease.
Graph showing the results of gel filtration using 200, Figure 2 is a graph showing the optimal pH distribution of serine protease, Figure 3 is a graph showing the optimal pH distribution of serine protease.
The figure is a graph showing the optimal temperature distribution of serine protease.
FIG. 4 is a graph showing the effect of serine protease in each solution on the contraction width of rat smooth muscle, and FIG. 5 is a graph showing the effect of serine protease on the KK- system in human plasma. Figure 2 Figure 3 pH Temperature (0C) Molecular weight (x104) 20 40 Fraction number (1,8ml'y:, =)) Figure 4

Claims (1)

[Claims] 1. A molecular weight of approximately 30,000 having the following physical and chemical properties:
A serine protease that activates the kallikrene-kinin system in human plasma. (1) Serine protease whose amino acid composition analysis value by hydrochloric acid hydrolysis method is as follows. Asx10.7Thr3.4 Ser9.4Glx10.7 Gly12.0Ala6.7 Val9.91/2Cys3.0 Met1.3Ile6.5 Leu5.8Tyr4.8 Phe0.9Lys6.0 His1.3Arg3.0 Pro3.2Trp1. 3 (2) 11th from NH_2 terminal and 2nd from 13th
A serine protease whose amino acid sequence up to position 0 is represented by formula (1) and formula (2). NH_2-terminal expression (1) [gene sequence available] NH_2-terminal expression (2) [gene sequence available] 2.
Affinity chromatography that specifically acts on the serine protease in an unpurified aqueous solution containing the serine protease according to claim 1, which is extracted from a rearing medium of the serine protease (des\farinae\), is used at least in the step of purifying the serine protease. A method for purifying the serine protease, characterized in that: