JP2002300886A - Stable recombinant human chymase, method for producing the same, and screening method by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a recombinant human chymase having such stability as higher than that of natural-type chymase and capable of being acquired in a large amount by means of gene recombination, so that research and development concerning the chymase are promoted by using the same. SOLUTION: This recombinant human chymase is obtained from a host infected with a recombinant baculovirus into which a human chymase-encoding gene is integrated, or obtained from cultured cells derived from the host, and has the following properties (1) to (3): (1) its chymase activity is not deactivated at 37 deg.C of the physiological temperature; (2) its action is completely inhibited by α-antitrypsin or chymostantin, but never inhibited by leupeptin, nor aprotinin, nor EDTA (ethylenediaminetetraacetic acid) at all; and (3) it exhibits a single band when subjected to electrophoresis after purified. A method for producing the same, a method for screening a substance which inhibits the action of the human chymase by using the same, and a method for evaluating and testing a test medicament by using the same are provided, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a stable recombinant human chymase, a method for producing the same, a method for screening a substance inhibiting the action of chymase using the same, and a method for evaluating and testing a test drug using the same. The recombinant human chymase obtained according to the present invention is a therapeutic agent for avoiding organ damage and protecting the organ, in particular, a therapeutic agent and a chimase for heart disease and hypertension or related diseases as the most studied diseases. It is used for screening of therapeutic drugs for allergic diseases involved. It is also useful for elucidating the pathological mechanism of chymase, and is used for diagnosis of pathological conditions and development of new therapeutic methods. Furthermore, it is used for the development of a recombinant human chymase preparation which applies the pharmacological activity of chymase itself to the treatment of skin diseases and the like.

[0002]

2. Description of the Related Art Human chymase is a type of serine protease found in secretory granules of mast cells, and its role is to produce angiotensin II (hereinafter abbreviated as "Ang II") or endothelin I (1-31). (Hereinafter abbreviated as “ETI (1-31)”) is known to be involved in production. Recently, other roles include extracellular matrix remodeling (activation of matrix metalloproteinases 1, 3, 9), network with cytokines (interleukin-1β activation, tumor activating factor (Tumor Growth Factor) β -1 secretion) and involvement in immunity. Therefore, the inhibitor to chymase is AngII or ETI (1-31)
Vascular lesions (PTCA)
Restenosis after operation, intimal hyperplasia after bypass graft), hypertension, cardiac hypertrophy, myocardial infarction, arteriosclerosis, heart and cardiovascular diseases such as diabetic and non-diabetic renal diseases, solid tumors and chymase It is expected to be a therapeutic drug for allergic diseases such as rheumatoid arthritis, keloids, psoriasis, and atopic dermatitis.

However, the conventional supply of chymase having enzymatic activity has been provided mainly by extraction from monkey myocardial tissue, not from humans. However, the supply of chymase from monkey myocardial tissue is not high and the amount recovered is very small, which has hindered the progress of chymase research. In addition, there are species differences in chymase, and humans, monkeys, and dogs have α chymase (angiotensin I (hereinafter, “Ang”).
I)), whereas rodents have several isoforms of chymase, whereas β chymase, which is predominantly present in rodents, produces AngII from AngI. Without this, it was found that an inactive fragment was generated. In order to clarify a pathological condition involving chymase from such characteristics, it is desirable to demonstrate using chymase.

[0004] However, natural human chymase is generally used for cell culture in screening tests and the like.
Under the condition of about ° C., as can be seen from the fact that it is decomposed in several hours, it is unstable to heat and is not homogeneous, so that it is not recognized as a single band in electrophoresis. Therefore, it has been difficult to use it for experiments to elucidate the pathological conditions involving chymase and for screening tests to find drugs that inhibit the function of human chymase.

On the other hand, a method for producing a recombinant human chymase using genetic engineering techniques has been reported.
In one cell or an expression system using Escherichia coli, the produced recombinant human chymase becomes insoluble in the cells,
Solubilization was required, which resulted in very low end product yields (eg, Husain).
[J. Biol. Chem., 268 (32); pp. 24318-24322 (199
3)], Wang et al. [Biol. Chem. Hoppe-Seyler., 37
6; pp681-684 (1995)]).

[0006] Production of recombinant human chymase has been attempted by using an insect culture cell-baculovirus system as a host (Walls et al. [Eur. J. BioCh.
em., 253; pp 300-308 (1998)] and Schmit et al. [Biol. Chem., 379; pp 167-174 (1998)].

However, the recombinant human chymase obtained by the above-described insect culture cell-baculovirus expression system is relatively unstable, and as a result, a sufficient amount of chymase has not been obtained. No comparison was made between human chymase and native human chymase.

[0008]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to obtain a large amount of recombinant human chymase having higher stability than natural human chymase by means of gene recombination, and to use the chimerase by utilizing this. To provide a method of screening for a substance that inhibits ATP and a method of evaluating and testing a drug. It is also to assist in elucidating the pathological mechanism of chymase, and to promote pathological diagnosis and the development of new treatments. Another object is to utilize the pharmacological activity of chymase itself for the development of a recombinant human chymase preparation which is applied to treatment.

[0009]

Means for Solving the Problems The present inventors, instead of expressing recombinant human chymase as an active form from the beginning, insert a peptide region cleaved by enterokinase between a signal peptide and a human chymase gene. First, an inactive recombinant human chymase containing the peptide region was produced. This prevented a decrease in yield due to autolysis. After purifying the inactive recombinant human chymase produced, the extra peptide portion is cleaved and removed by enterokinase and converted to a recombinant human chymase with chymase activity, thereby obtaining a recombinant human chymase at a high recovery rate. I found what I could do. In addition, they have found that recombinant human chymase is efficiently secreted by using a signal peptide of a secretory protein derived from a production host as the signal peptide.

[0010] The obtained recombinant human chymase is different from natural human chymase in several points such as high heat stability, and can be advantageously used in a method for screening a substance that inhibits the function of chymase. Thus, the present invention has been completed.

That is, an object of the present invention is to provide a recombinant human chymase having the following properties and obtained from a host or host cell infected with a recombinant virus incorporating a gene encoding human chymase. (1) Chymase activity is not lost at a physiological temperature of 37 ° C. (2) completely inhibited by α-antitrypsin, chymostatin, leupeptin, aprotinin and ED
Not inhibited at all by TA. (3) shows a single band in electrophoresis after purification.

It is another object of the present invention to infect a host or host cell with a recombinant virus incorporating a gene encoding a protein secretory signal peptide, a gene encoding an enterokinase cleavage sequence, and a gene encoding human chymase. After incubating the recombinant virus, obtaining an inactive recombinant human chymase from the host or the host cell culture supernatant, and converting it to an active form using enterokinase. The present invention provides a method for producing human chymase.

[0013] Still another object of the present invention is to provide a method for screening a substance that inhibits the action of chymase using the above-mentioned recombinant human chymase.

Still another object of the present invention is to provide a test method for evaluating a test drug utilizing the above-mentioned recombinant human chymase.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The recombinant human chymase of the present invention can be produced by the following procedure. That is,
Construct a recombinant virus incorporating the gene encoding the protein secretion signal peptide of the host, the gene encoding the enterokinase cleavage sequence, and the gene encoding human chymase, and infecting the host or host cell after growing the recombinant virus After the recombinant virus is propagated, an inactive recombinant human chymase is obtained from the host or the host cell culture supernatant, purified, and then converted to an active form using enterokinase. it can.

The recombinant virus used in the present invention is, for example, a gene encoding a secretory protein signal peptide of the host and a gene encoding an enterokinase cleavage sequence in a baculovirus such as silkworm nuclear polyhedrosis virus (BmNPV). And a gene encoding human chymase.

In the present invention, the gene encoding a host protein secretion signal to be incorporated into the recombinant virus (hereinafter referred to as "signal peptide gene") includes:
For example, when the host is a silkworm, a gene encoding a signal peptide of the silkworm 30K protein can be mentioned. This gene is described in, for example, Sakai et al. (Biochem. Bio
phys. Acta., 949; pp. 224-232, (1988)) and can be obtained based on this.

The gene encoding human chymase (hereinafter referred to as "human chymase gene") has been reported by Takai et al. (FEBS. Let., 412, pp86-90, (1997)). Can be obtained by performing RT-PCR based on

Further, the addition of a gene encoding an enterokinase cleavage sequence to the human chymase gene may be performed by providing an enterokinase cleavage site in the primer sequence,
It is possible to use a cloning vector such as pBluBacHis2B (INVITROGEN).

Each of these genes is preferably inserted downstream of the viral polyhedrin promoter in the order of a signal peptide gene, a gene encoding an enterokinase cleavage sequence, and a human chymase gene.

The binding of each of these genes can be carried out by obtaining the above genes by a conventional method, and using appropriate primers if necessary.
After digestion with a restriction enzyme, ligation may be performed using a ligation kit. Specifically, cDNA amplified by PCR using primers having appropriate restriction enzyme sites
The fragment can be digested with a restriction enzyme, and then mixed with a transfer vector previously digested with the same restriction enzyme and a ligation reagent.

The amplification, acquisition, or incorporation of the human chymase gene into a virus can be carried out by a conventional method. For example, amplification or acquisition of the human chymase gene is performed by PCR using appropriate primers.
Reaction may be carried out, and integration into the virus may be performed according to Maeda et al. [Annu. Rev. Entomol., 34; pp351-372 (19
89)].

The recombinant virus thus obtained is then infected to a host and propagated. The host used may be the host of the virus itself or a cultured cell from the host. Specifically, as a host used for the production of the recombinant human chymase of the present invention, if the recombinant virus is based on BmNPV, a silkworm body, a pupa or a silkworm culture cell is used. You.

Known methods can be used for the method of infecting the host or cultured cells of the host with the recombinant virus, and the method of propagation after infection. For example, in the case where silkworm bodies are used as a host, the recombinant virus can be taken into these by oral infection or infection by injection.

As a method for separating and collecting the inactive recombinant human chymase produced by the infection and propagation method from the host or cultured cells of the host as described above, known methods can be used. . For example, when a silkworm body is used as a host, it can be performed as follows. That is, first, a body fluid is collected from a silkworm that has been infected and bred with the recombinant virus, and purified.

Purification of the inactive recombinant human chymase from the body fluid of the silkworm is preferably performed by column chromatography in the order of a cation exchange column and then a heparin column. Among them, the cation exchange column chromatography is preferably performed at a relatively high pH (for example, around pH 7.8) because it is easy to separate from other miscellaneous proteins in the silkworm body fluid. There is a method using an anion exchange column, gel filtration column, or heparin column as the primary column. However, in this method, since the amount of other miscellaneous proteins to be mixed is large, inactive recombinant Isolation of human chymase is difficult and is not a preferred method.

The inactive recombinant human chymase thus obtained has an enterokinase cleavage sequence added thereto, and thus is subjected to an activation treatment. This treatment is carried out by using enterokinase and, for example, incubating at a temperature of about 25 ° C. for 6 hours or more.

By performing a separation treatment using a column after the above treatment, contaminating enterokinase-cleaved peptide and enterokinase can be removed, and only a recombinant human chymase having the desired chymase activity can be obtained. . Heparin column chromatography is preferably used to remove the cleaved enterokinase-cleaved peptide and enterokinase and to isolate the recombinant human chymase with chymase activity.

The recombinant human chymase thus obtained has almost the same physical properties as the natural human chymase, but is clearly different in that the stability is high.
For example, under general animal culture cell culture conditions,
% FBS containing MEM medium (minimum essential mediu
m; Sigma) and incubation in a thermostat at 37 ° C., the native human chymase immediately lost chymase activity, but the recombinant human chymase retained chymase activity for several days.

[0030]

The expression systems conventionally used for the production of recombinant human chymase include Escherichia coli, Bacillus subtilis, yeast and baculovirus. To obtain recombinant human chymase from these expression systems, ,
There were the above-mentioned problems.

On the other hand, in the present invention, a host peptide is used as a signal peptide at the same time as using a baculovirus expression system, thereby solving the above problems and obtaining a large amount of recombinant human chymase. Was made possible.

Furthermore, the recombinant human chymase obtained according to the present invention has a very high stability as compared with the natural human chymase. This is a very advantageous property compared to.

According to the present invention, a recombinant human chymase can be produced in large quantities and advantageously. The obtained recombinant human chymase has higher stability under general reaction conditions and culture conditions of animal cultured cells than natural human chymase, and thus is a therapeutic drug for diseases caused by human chymase. Can be used as a screening reagent.

That is, the recombinant human chymase of the present invention and a test drug are injected into a medium for culturing cells and tissue fragments sensitive to human chymase, and the resulting cell change is detected. It can be screened whether or not the test drug has the activity of inhibiting the activity of human chymase by comparing the change of the cells in the medium into which only human chymase has been injected. Further, the method can be applied to a drug evaluation test using this method, a drug evaluation test using a living body, and the like.

[0035] Recombinant human chymase plays an important role in elucidating the pathological mechanism of chymase, and may be used for diagnosis of pathological conditions and development of new therapeutic methods in the future. Furthermore, elucidation of the pharmacological activity of chymase itself has progressed, and it can be applied to the development of a recombinant human chymase drug utilizing chymase itself. According to this production method, since a large amount of recombinant human chymase is obtained, it is possible to cope with the three-dimensional structure analysis of human chymase, and drug design using three-dimensional structure analysis data becomes possible.

[0036]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention.

Example 1 An inactive recombinant human chymase having an enterokinase cleavage sequence (DDDDK) added to its N-terminus (hereinafter referred to as EK-
Preparation of Virus Expressing Chymase) (1) Cloning of Human Chymase cDNA (a) Acquisition of Human Chymase cDNA The human chymase cDNA was obtained from a cDNA synthesis kit (Gibco
(BML) using the RT-PCR method. First, using 4 μg of total RNA extracted from human blood vessels,
The following sense and antisense primers were designed with reference to the previously reported base sequence of human chymase (Takai et al. [FEBS. Let., 412, pp86-90, (1997)]) and 1 minute at 94 ° C, 1 minute at 63 ° C, 7
The RT-PCR reaction was performed 40 times at 2 ° C. for 1 minute to amplify human chymase cDNA.

Sequence of sense primer: 5'-TTGCTCAGGCAGCCTCTCTG-3 '

Sequence of antisense primer: 5'-TTCAGGCTGGCTCAGGATCC-3 '

(B) Construction of a cloning vector in which an enterokinase cleavage site has been added to the N-terminus of human chymase: First, using the human chymase cDNA obtained above as a template, the previously reported base sequence of human chymase (Takai et al. [FEBS Let., 412, pp86-90, (1997)], a sense (His / EK-CHY) and an antisense primer (CHY-salI) having the following enterokinase cleavage sites were designed. PCR reaction was carried out using the same, and EK-chymase cD
NA was obtained. The obtained EK-chymase cDNA was treated with restriction enzymes SphI and SalI, and then subcloned between SphI and SalI of cloning vector pQE31 (QIAGEN) to construct pQE / EK-CHY.

Sense primer (His / EK-CH)
Sequence of Y): 5'-CCGCATGCCGATGACGATGACAAAATCATCGGGGGCACAGAATG-3 '

An antisense primer (CHY-Sal
Sequence of I): 5'-GAGCAGCTGAATTAAACGGACGACGTCCTAG-3 '

(2) Construction of transfer vector having secretory protein 30K protein secretion signal sequence derived from silkworm: Nucleotide sequence of secretion signal of “30K protein” which is a secretory protein derived from silkworm (Sakai et al., 19)
88: Biochim. Biophys. Acta 949, 224-232)
Oligonucleotides for the sense and antisense strands were synthesized.

These were treated with 9 mM in the presence of 50 mM NaCl.
By annealing at 5 ° C. for 55 minutes, 55 ° C. for 15 minutes, and 37 ° C. for 15 minutes, a signal oligo DNA having a 30K protein secretory signal sequence was obtained. This signal oligo DNA was inserted between BglII and SacI of a pYNG transfer vector (Katakura Industries) by a ligation reaction (Ligation Kit, TaKaRa) to construct a transfer vector (pYNGsig) having a 30K protein secretion signal sequence.

(3) Construction of transfer vector into which sig-EK-chymase gene was introduced: As shown in FIG.
In order to introduce chymase cDNA, it was once subcloned into a pTZ8 cassette vector (Katakura Industry Co., Ltd.), and a restriction enzyme site was newly added. That is, pQE / E
K-CHY was digested with restriction enzymes SphI and SalI to cut out an EK-chymase cDNA fragment, which was
It was inserted into the SphI-SalI restriction enzyme site of Z8 (pTZ8EKChym).

Next, after digesting pTZ8EKChym with a restriction enzyme XbaI, a blunting kit (Bl
unting Kit; TaKaRa), and further digested with restriction enzyme EcoRI to obtain EK-chymase c.
DNA was cut out. This is called Sma of pYNGsig.
By inserting between I-EcoRI, a 30K protein secretion signal sequence, E
A transfer vector (pYNGsigEKChym) into which genes were introduced in the order of the K-chymase sequence was obtained (FIG. 1). FIG. 2 shows the 30K protein secretion signal and the sequence between EK and chymase of the transfer vector.

(4) Preparation of sig-EK-Chymase Genetically Recombinant Baculovirus Recombinant baculovirus was prepared according to Maeda et al.
al., Annu. Rev. Entomol., 34; pp351-372 (1989))
The method was modified. That is, first, pYNGsi
The gEKChym plasmid DNA was purified using a plasmid purification kit (QIAGEN). Next, linearized baculovirus (strain CPd: Japanese Patent No. 3,090,586, US Pat. No. 5,753,2)
No. 20) and purified pYNGsigEKChy
m plasmid DNA was mixed, and BmN cells (Maeda et al., Ann.) were mixed with a lipofectin reagent (Gibco BML).
u. Rev. Entomol., 34; pp 351-372 (1989)).

Screening of recombinant baculovirus was performed by limiting dilution using a 96-well plate (Maeda et al.
l., Annu. Rev. Entomol., 34; pp351-372 (1989)), and viruses that did not form a polyhedron were selected. Next, the virus-infected cells were collected and EK-chymase cDN
Using the A probe, a recombinant virus in which the expression of EK-chymase mRNA was found and the EK-chymase gene was introduced into the virus genome was selected and cloned by Southern blot analysis and Northern blot analysis ( CPdsigEKChym virus).

Example 2 Production and Purification of Recombinant Human Chymase in Silkworms: (1) Production of EK-Chymase in Silkworm Larvae CPdsigEKChym virus was inoculated into silkworms on the 5th day and the 5th day of virus infection. The body fluid was collected. The collected body fluid was centrifuged at 25,000 rpm for 60 minutes, and the supernatant was collected to remove the mixed tissue pieces and the like. The obtained supernatant was added to a three-fold amount of 50 mM sodium phosphate (pH
7.8) (Buffer A) was added to prepare a body fluid containing EK-chymase.

(2) Purification of recombinant human chymase expressed in silkworm body fluid: (a) Purification of EK-chymase by column chromatography Equilibration of Sp Toyopearl cation exchange column (Tosoh) against buffer A After that, a silkworm body fluid containing EK-chymase was added for adsorption. Next, 0.2M N
After washing with buffer A containing aCl, the salt concentration was reduced to 0.
EK-Chymase was eluted by raising to 5M NaCl ((A) in FIG. 3). The fraction containing EK-chymase was prepared by adding an equal volume of 50 mM Tris-HCl (pH 8.0) (buffer B) to a final NaCl concentration of 250 mM.

Subsequently, a heparin affinity column (HiTrap Heparin, Pharmacia) was equilibrated against buffer B, and the fraction containing EK-chymase recovered from the cation exchange column was added and adsorbed. After washing with buffer B first, it was washed again with buffer B containing 0.4 M NaCl. Elution of EK-chymase was carried out by adjusting the salt concentration in buffer B to 0.4 to 1.0 M NaC.
1 by applying a linear gradient of EK-
Chymase was eluted under a salt concentration condition of 0.5 M NaCl ((B) in FIG. 3). In addition, all the steps related to the column operation were performed at 4 ° C.

(B) EK-Chymase activation treatment 45 units of enterokinase (Funakoshi) were added per 1 μg of EK-Chymase obtained in (a) above, and
Incubation for 6 hours or more at
An enterokinase-cleaved peptide attached to the N-terminus of chymase was removed to obtain an active recombinant human chymase having enzymatic activity (hereinafter, abbreviated as recombinant human chymase).
FIG. 4 shows a state in which EK-chymase changes into a recombinant human chymase over time by enterokinase treatment.

(C) Purification of Recombinant Human Chymase In order to remove the enterokinase-cleaved peptide and enterokinase from the recombinant human chymase obtained in (b) above, affinity purification using a heparin column was performed again in the same manner as in (a). Eluting the recombinant human chymase with a linear gradient of 0.4 to 1.0 M NaCl,
It was isolated ((C) in FIG. 3). In addition, all the steps related to the column operation were performed at 4 ° C.

Table 1 shows the EK-chymase obtained in (a) and the recombinant human chymase obtained in (c).
The results obtained by measuring chymotrypsin activity, specific activity and the like as the obtained capacity, protein concentration, total protein amount and chymase activity are shown together.

[0055]

[Table 1]

(3) Confirmation of Recombinant Human Chymase in Each Purification Step Detection of recombinant human chymase in each purification step was performed by SDS.
-Performed by polyacrylamide electrophoresis pattern and chymase activity assay.

(A) Detection of SDS-polyacrylamide electrophoresis pattern by silver staining Samples fractionated in each purification step of Example 2- (2) were subjected to SDS-polyacrylamide electrophoresis (Laemmli, Nature 227, pp68).
0, (1970)). 1 for SDS-PAGE gel
The sample was used for electrophoresis at a constant current (20 mA) together with a molecular weight marker. After the electrophoresis, the proteins were confirmed by a silver staining kit (Silver Kit Kit Wako; Wako Pure Chemical Industries), and the degree of purification in each purification step was confirmed (FIG. 5).

(B) Chymase activity measurement method The measurement of the recombinant human chymase activity in each purification step of Example 2- (2) was performed by measuring the chymotrypsin activity in the solution after the activation treatment. . That is,
The fractionated sample from each purification step was treated with an appropriate amount of enterokinase (Funakoshi), and then the fluorescent substrate N-
Succinyl-alanyl-alanyl-phenylalanyl-
Prolyl-7-amido-4-methylcoumarin (hereinafter referred to as S
uc-Ala-Ala-Pro-Phe-MCA; Peptide Research Laboratories) containing a final concentration of 80 μM (0.1 M Tris-HCl (pH 8.0), 0.1 M).
5M NaCl) at 37 ° C. for 30 minutes, and the amount of released 7-amino-4-alanyl-methyl coumarin (hereinafter abbreviated as MCA) was measured using a fluorometer at an excitation wavelength of 3 μm.
Chymase activity was calculated by measuring the fluorescence intensity under the conditions of 80 nm and a fluorescence wavelength of 460 nm. In addition, 1
The activity of decomposing 1 μmole of the substrate per minute was defined as 1 unit.

Purification proceeded by fractionating fractions in which chymase activity was present at significant levels in the solution,
By examining the results together with the results of silver staining, it was confirmed that the recombinant human chymase was isolated.

Test Example 1 Km Value and Molar Activity of Recombinant Human Chymase Activity The Km value and molar activity of the obtained recombinant human chymase were determined from the change in activity with respect to the change in substrate concentration when AngI was used as a substrate. That is, from AngI to AngII
Peptide fragment His that is released by the conversion reaction into
-Leu (Wako Pure Chemical Industries) was converted to a fluorescent derivative with o-phthalaldehyde (Wako Pure Chemical Industries), and the fluorescence intensity was calculated by measuring the fluorescence intensity. The substrate solution was prepared by dissolving AngI (Sigma) in distilled water and further dissolving 0.3 in distilled water.
1, 0.63, 1.3, 2.5, 5.0, 10, and 20 mM
It was prepared so that The enzyme solution was prepared by diluting the recombinant human chymase obtained in Example 2 (c) with distilled water to obtain 22 ng of the enzyme solution.
/ Ml.

Next, as a pretreatment for the enzymatic reaction, a reaction solution (20 mM Tris-HCl (pH 8.0), 0.5
M KCl, 0.1% Triton X-100) 80μ
Then, 10 μl of the enzyme solution was added to the mixture and incubated at 37 ° C. for 10 minutes. Thereafter, 10 μl of the further prepared substrate solution
Was added and reacted at 37 ° C. for 120 minutes. The enzymatic reaction was stopped by adding 150 μl of ice-cooled 15% trichloroacetic acid. Thereafter, the mixture was centrifuged at 4 ° C. and 16000 rpm for 10 minutes, and 200 μl of the supernatant was mixed with a fluorescence inducing reaction solution (1% o-phthalaldehyde, 0.2 M NaO 2).
H) The mixture was mixed with 200 μl, and a fluorescence inducing reaction was performed at room temperature for 10 minutes. Termination of the fluorescence induction reaction was carried out by 6M HC.
1 by adding 200 μl. The fluorescence intensity was measured with a fluorometer under the conditions of an excitation wavelength of 350 nm and a fluorescence wavelength of 455 nm. Further, His-L is used as a standard substance.
A calibration curve was prepared using eu, and the amount of substrate degradation was quantified.

As a result, both reciprocal plots of the substrate concentration and the chymase activity showed a straight line, confirming that the enzymatic reaction of the recombinant human chymase followed the Michaelis-Menten type rate process (FIG. 6). Then, the Km value and the molar activity of the recombinant human chymase were calculated by fitting to the Michaelis-Menten equation by the nonlinear least squares method, and were found to be 53.7 μM and 78.7 / s, respectively. These values are based on Takai et al. [Clinica Climica Acta, 265; pp13-20 (199
7)] (Km
(67 μM, molar activity 65 / s).

Test Example 2 Examination of optimum pH for enzyme activity: An was performed in the same manner as in Test Example 1 above.
The assay was performed using gI as a substrate. However, the buffer used for the reaction solution was 20 mM Tris-HCl (pH 8.
0) to 20 mM Britton-R
obinson) buffer (pH 6.0 to pH 10.0)
Was changed. Chymase activity is from pH 6.0 to pH 10.0
The intervals were measured at intervals of 0.5 in nine steps.

As a result, the recombinant human chymase had a pH
It shows strong activity in the range of 7.0 to pH 9.0 and pH 8.0.
The maximum activity was shown at 0. These values are based on Takai et al. [Cl
inicaClimica Acta, 265; pp13-20 (1997)], which was almost equivalent to the optimal pH (pH 7.5 to pH 9.0) of natural human chymase (FIG. 7).

Test Example 3 Examination of substrate specificity: AngI, AngII (Peptide Research Institute), Substance P (Sigma), Bradykinin (Sigma), Neurotensin (Sigma) and Big Endothelin 1 (hereinafter “bigET1”) Abbreviated)
The enzyme reaction specificity of the recombinant human chymase was examined based on the presence or absence of (Sigma) hydrolysis. The substrate solution is Ang
I, AngII, substance P, bradykinin, and neurotensin were each dissolved in distilled water and adjusted to 10 mM. The bigET1 was dissolved in distilled water,
It was adjusted to 1 mM and used as a substrate solution. The enzyme solution was prepared in Example 2.
The recombinant human chymase obtained in (c) was diluted with distilled water to adjust to 2.2 μg / ml.

Next, the reaction solution (20 mM Tris-H)
Cl (pH 8.0), 0.5 M KCl, 0.1% Tri
Ton X-100) was added to 10 μl of the enzyme solution and 10 μl of the prepared substrate solution, and reacted at 37 ° C. for 4 hours. The enzymatic reaction was stopped by adding 150 μl of ice-cold methanol. Thereafter, the mixture was centrifuged at 4 ° C. and 12000 rpm for 10 minutes, and 50 μl of the supernatant was collected.
Analysis was performed using an LC system (WATERS).

AngI, AngII, substance P,
HPLC analysis using bradykinin and neurotensin as substrates showed a detection column Puresil 5 μC18 12018 equilibrated with 10 mM phosphate buffer (pH 3.8) -methanol (90:10).
(φ4.6 × 150 mM) was injected with 50 μl of the supernatant, and eluted by a method in which the methanol concentration was increased by a linear concentration gradient of 10-90%. The eluted material was analyzed by ultraviolet absorption (measurement wavelength:
215 nm).

A peak was identified from the retention time at the time of injecting a standard (0.4 mM) of each substrate, and the peak height at this time was set to 1
00%, the height of the substrate peak after the reaction is less than 20%,
20% to less than 70%, 70% to less than 90% and 90%
%, The presence or absence of a reaction was evaluated as ++, +, ±, and-, respectively.

The HPLC analysis using bigET1 as a substrate showed that the detection column pure acyl 5 μC18 120 ° (φ4.6 × 150) equilibrated with 0.1% trifluoroacetic acid-acetonitrile (80:20).
mM), and eluted with a method in which the concentration gradient of the starting solution acetonitrile was linearly increased to 20-35%. The eluate was detected by ultraviolet absorption (measurement wavelength: 215 nm). Table 2 shows the results.

[0070]

[Table 2]

As shown in Table 2, the recombinant human chymase digested AngI, neurotensin, and bigET1, but did not digest AngII, substance P, and bradykinin, similarly to the natural human chymase. Also,
The reaction product peaks of AngI and bigET1 by the recombinant human chymase and the natural human chymase coincided with the peaks of AngII and ET1 (1-31), respectively, and it was confirmed that both decompose the substrate at the same cleavage site. .

Test Example 4 Examination of sensitivity to various inhibitors: The sensitivity of human chymase to various inhibitors was evaluated by the residual activity of chymase upon addition of various inhibitors. Chymase activity is Suc-Al
The amount of MCA (Peptide Institute) released using a-Ala-Pro-Phe-MCA as a substrate was measured by spectrophotometric quantification. First, the fluorescent substrate Suc-Ala-
Ala-Pro-Phe-MCA was dissolved in DMSO to prepare a 10 mM substrate solution. In addition, various inhibitors include chymostatin (Peptide Research Institute) in DMSO, leupeptin (Sigma) and α-antitrypsin (CHEMICON IN).
TERNATIONAL, Inc.), aprotinin (BIOSYS SA), soybean trypsin inhibitor (hereinafter abbreviated as "SBTI";
Worthington Biochem. Co.) and EDTA were dissolved in distilled water to give 1 mM, 1 mg / ml, 1 mg / ml and 5 mg, respectively.
The concentrations were adjusted to mg / ml, 10 mM, and 1 mg / ml. The enzyme solution was prepared by diluting the recombinant human chymase obtained in Example 2 (c) with distilled water to a concentration of 2.2 μg / ml.

Next, as a pretreatment for the enzymatic reaction, a reaction solution (20 mM Tris-HCl (pH 8.0), 0.5 M
KCl, 0.1% Triton X-100) 170μ
Then, 10 µl of the enzyme solution and 10 µl of the prepared various inhibitors were added to the mixture, and the mixture was incubated at 37 ° C for 10 minutes. Thereafter, 10 μl of a substrate solution was further added and reacted at 37 ° C. for 120 minutes. The enzymatic reaction was stopped by adding 800 μl of 50% acetic acid. Thereafter, the mixture was centrifuged at 4 ° C. and 16,000 rpm for 10 minutes, and 900 μl of the supernatant was added to 1.1 m of distilled water.
After dilution with 1, the excitation wavelength is 380 n using a fluorometer.
m, the fluorescence intensity was measured under the conditions of a fluorescence wavelength of 460 nm.
Furthermore, a calibration curve was created using MCA as a standard substance,
The amount of substrate degradation was quantified.

As a result, the chymase activity of the recombinant human chymase was inhibited by chymostatin, α-antitrypsin and SBTI as in the case of the natural human chymase.
On the other hand, chymase activity was not inhibited by leupeptin, aprotinin, and EDTA (FIG. 8).

Test Example 5 Examination of stability in various preservation solutions: recombinant human chymase solution and natural human chymase solution and 1 mg / ml BS
A containing PBS solution or animal cell culture medium (5% FBS
Were mixed in a constant temperature bath at 37 ° C., sampling was performed with time, and chymase activity was measured. The measurement of chymase activity was in accordance with Example 2 (3) (b).

As a result, the recombinant human chymase was more stable in these solutions than the natural human chymase.
In particular, in tests in animal cell culture, native human chymase lost chymase activity within hours, whereas recombinant human chymase retained chymase activity for several days (FIG. 9).

[0077]

According to the present invention, a large amount of recombinant human chymase can be produced. Furthermore, the recombinant human chymase obtained by the present invention has the same substrate specificity as the natural human chymase and is stable in a solution. Thus, screening of substances that inhibit the function of human chymase and drug evaluation tests can be performed.
And, from the found human chymase inhibitor, a disease caused by human chymase, specifically, restenosis after PTCA, which is the best studied disease, and intimal hyperplasia after bypass graft, hypertension,
Cardiac and cardiovascular diseases such as cardiac hypertrophy, myocardial infarction, arteriosclerosis, diabetic and non-diabetic renal diseases, solid tumors and allergic diseases involving chymase (eg, rheumatoid arthritis, keloids, psoriasis, atopy) Dermatitis) can be developed. In addition, recombinant human chymase plays an important role in elucidating the pathological mechanism of chymase, and can be used for diagnosis of pathological conditions and development of new therapeutic methods in the future. Furthermore, it is also possible to develop a recombinant human chymase preparation or the like in which the pharmacological activity of chymase itself is applied to the treatment of skin diseases and the like.

[0078]

[Sequence List] <110> katakura Industries Co., Ltd. TOA EIYO LTD. <120> Stable recombinant human chymase, process for the preparation of them, and method for screening with them <130> 9910106 <140> <141> < 160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 789 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (1) .. (786) <223> Sig- EK-Chymase <220> <221> mat_peptide <222> (109) .. (786) <223> mature-Chymase <220> <221> gene <222> (94) .. (108) <223> enterokinase recognition site <220> <221> sig_peptide <222> (1) .. (57) <223> signal sequence originated in 30K-protein from silkworm hemolymph <220> <221> unsure <222> (58) .. (93) <223> linker gene sequence <220> <221> gene <222> (58) .. (768) <223> EK-Chymase <300> <302> Cloning of the gene and cDNA for human heart chymase <303> J Biol. Chem. <304> 266 <306> 17173-17179 <307> 1991 <400> 1 atg aga ctg act ttg ttt gcc ttc gtc ctc gcc gtg tgt gcg ctg gct 48 Met Arg Leu Thr Leu Phe Ala Phe Val Leu Ala Val Cys Ala Leu Ala -35 -30 -25 tct aac gcc aga gc t ccc cta gat atc cat gca agc ttg cat gcc gat 96 Ser Asn Ala Arg Ala Pro Leu Asp Ile His Ala Ser Leu His Ala Asp -20 -15 -10 -5 gac gat gac aaa atc atc ggg ggc aca gaa tgc aag cca cat tcc cgc 144 Asp Asp Asp Lys Ile Ile Gly Gly Thr Glu Cys Lys Pro His Ser Arg -1 1 5 10 ccc tac atg gcc tac ctg gaa att gta act tcc aac ggt ccc tca aaa 192 Pro Tyr Met Ala Tyr Leu Glu Ile Val Thr Ser Asn Gly Pro Ser Lys 15 20 25 ttt tgt ggt ggt ttc ctt ata aga cgg aac ttt gtg ctg acg gct gct 240 Phe Cys Gly Gly Phe Leu Ile Arg Arg Asn Phe Val Leu Thr Ala Ala 30 35 40 cat tgt gca gga agg tct ata aca gtc acc ctt gga gcc cat aac ata 288 His Cys Ala Gly Arg Ser Ile Thr Val Thr Leu Gly Ala His Asn Ile 45 50 55 60 aca gag gaa gaa gac aca tgg cag aag ctt gag gtt ata aag caa ttc 336 Thr Glu Glu Glu Asp Thr Trp Gln Lys Leu Glu Val Ile Lys Gln Phe 65 70 75 cgt cat cca aaa tat aac act tct act ctt cac cac gat atc atg tta 384 Arg His Pro Lys Tyr Asn Thr Ser Thr Leu His His Asp Ile Met Leu 80 85 90 cta aag ttg aag gag a aa gcc agc ctg acc ctg gct gtg ggg aca ctc 432 Leu Lys Leu Lys Glu Lys Ala Ser Leu Thr Leu Ala Val Gly Thr Leu 95 100 105 ccc ttc cca tca caa ttc aac ttt gtc cca cct ggg aga atg tgc cgg 480 Pro Pro Ser Gln Phe Asn Phe Val Pro Pro Gly Arg Met Cys Arg 110 115 120 gtg gct ggc tgg gga aga aca ggt gtg ttg aag ccg ggc tca gac act 528 Val Ala Gly Trp Gly Arg Thr Gly Val Leu Lys Pro Gly Ser Asp Thr 125 130 135 140 ctg caa gag gtg aag ctg aga ctc atg gat ccc cag gcc tgc agc cac 576 Leu Gln Glu Val Lys Leu Arg Leu Met Asp Pro Gln Ala Cys Ser His 145 150 155 ttc aga gac ttt gac cac aatg ctt cag tgt gtg ggc aat ccc agg 624 Phe Arg Asp Phe Asp His Asn Leu Gln Leu Cys Val Gly Asn Pro Arg 160 165 170 aag aca aaa tct gca ttt aag gga gac tct ggg ggc cct ctt ctg tgt 672 Lys Thr Lys Ser Ala Phe Lys Gly Asp Ser Gly Gly Pro Leu Leu Cys 175 180 185 gct ggg gtg gcc cag ggc atc gta tcc tat gga cgg tcg gat gca aag 720 Ala Gly Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Ser Asp Ala Lys 190 195 200 ccc cct gc t gtc ttc acc cga atc tcc cat tac cgg ccc tgg atc aac 768 Pro Pro Ala Val Phe Thr Arg Ile Ser His Tyr Arg Pro Trp Ile Asn 205 210 215 220 cag atc ctg cag gca aat taa 789 Gln Ile Leu Gln Ala Asn 225 <210> 2 <211> 262 <212> PRT <213> Artificial Sequence <400> 2 Met Arg Leu Thr Leu Phe Ala Phe Val Leu Ala Val Cys Ala Leu Ala -35 -30 -25 Ser Asn Ala Arg Ala Pro Leu Asp Ile His Ala Ser Leu His Ala Asp -20 -15 -10 -5 -5 Asp Asp Asp Lys Ile Ile Gly Gly Thr Glu Cys Lys Pro His Ser Arg -1 1 5 10 Pro Tyr Met Ala Tyr Leu Glu Ile Val Thr Ser Asn Gly Pro Ser Lys 15 20 25 Phe Cys Gly Gly Phe Leu Ile Arg Arg Asn Phe Val Leu Thr Ala Ala 30 35 40 His Cys Ala Gly Arg Ser Ile Thr Val Thr Leu Gly Ala His Asn Ile 45 50 55 60 Thr Glu Glu Glu Asp Thr Trp Gln Lys Leu Glu Val Ile Lys Gln Phe 65 70 75 Arg His Pro Lys Tyr Asn Thr Ser Thr Leu His His Asp Ile Met Leu 80 85 90 Leu Lys Leu Lys Glu Lys Ala Ser Leu Thr Leu Ala Val Gly Thr Leu 95 100 105 Pro Phe Pro Ser Gln Phe Asn Phe Val Pro Pro Gly Arg Met Cys Arg 110 115 120 Val Ala Gly Trp Gly Arg Thr Gly Val Leu Lys Pro Gly Ser Asp Thr 125 130 135 140 Leu Gln Glu Val Lys Leu Arg Leu Met Asp Pro Gln Ala Cys Ser His 145 150 155 Phe Arg Asp Phe Asp His Asn Leu Gln Leu Cys Val Gly Asn Pro Arg 160 165 170 Lys Thr Lys Ser Ala Phe Lys Gly Asp Ser Gly Gly Pro Leu Leu Cys 175 180 185 Ala Gly Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Ser Asp Ala Lys 190 195 200 Pro Pro Ala Val Phe Thr Arg Ile Ser His Tyr Arg Pro Trp Ile Asn 205 210 215 220 Gln Ile Leu Gln Ala Asn 225

[Brief description of the drawings]

FIG. 1 is a drawing showing the steps of constructing a transfer vector (pYNGsigEKChym) for introducing sig-EK-chymase DNA downstream of a polyhedrin promoter in the order of a 30K protein secretion signal sequence and an EK-chymase sequence.

FIG. 2. Transfer vector (pYNGsigE
1 is a drawing showing the 30K protein secretion signal of KChym) and the sequence between EK and chymase. In the figure, (I) is 30K
(II) shows an EK-chymase sequence, (a) shows a linker sequence, (b) shows an enterokinase cleavage sequence, and (c) shows a recombinant human chymase sequence.

FIG. 3 is a diagram showing elution curves in each purification step of columnar chromatography of recombinant human chymase from silkworm body fluid. In the figure, (A) is an elution curve diagram of EK-chymase using a cation exchange column, and (B) is
Elution curve diagram of EK-chymase by heparin column,
(C) shows the elution curve diagram of the recombinant human chymase using a heparin column.

FIG. 4 is an SDS-PAGE electrophoretogram showing the change over time in the activation of EK-chymase by enterokinase.

FIG. 5 shows S in each purification step of columnar chromatography of recombinant human chymase from silkworm body fluid.
It is an electrophoretogram of DS-PAGE.

FIG. 6 is a diagram showing a reciprocal plot of a measured value and a substrate concentration in an enzyme reaction using AngI of recombinant human chymase as a substrate (however, one unit is 1 unit).
Per minute, the synthetic substrate AngI is hydrolyzed, which is the μmol number of His-Leu produced).

FIG. 7 is a diagram showing the pH dependence of measured values in an enzyme reaction using AngI of recombinant human chymase as a substrate (however, 1 unit (unit) means that the synthetic substrate AngI is hydrolyzed per minute, His-Leu to be generated
Is the number of μmol).

FIG. 8 is a view showing a difference in sensitivity between a natural human chymase and a recombinant human chymase to various inhibitors (however, one unit is a fluorescent substrate Suc-Ala-Ala-Pro-Phe per minute). -Is the μmol of MCA produced by hydrolysis of MCA).

FIG. 9 is a diagram showing a comparison of the stability of chymase activity between a recombinant human chymase solution and a natural human chymase solution and a PBS solution containing 1 mg / ml BSA or an animal cell medium (however, one unit is 1 unit). Fluorescent substrate Suc-Ala-Ala-Pro-Ph per minute
e-MCA is hydrolyzed and μmol of MCA produced
Number). that's all

Continued on the front page (72) Inventor Akira Tatsui 1 Tanaka Tanaka, Yuno, Iizaka-cho, Fukushima City, Fukushima Prefecture (72) Inventor Miyazaki Mizuo 4-1-1, Tomookacho, Nagaokakyo-shi, Kyoto (72) Inventor Shinji Takai Osaka 1-12-12 Akutagawa-cho, Takatsuki-shi F-term (reference) 4B024 AA01 AA11 BA14 CA04 EA02 GA11 HA01 HA08 HA12 4B050 CC03 DD11 FF13C LL01 LL03 4B063 QA01 QA18 QQ20 QQ36 QR48 QR58 QS28 QX02 4B095 AA93A44A

Claims (10)

[Claims] A recombinant human chymase having the following properties and obtained from a host or host cell infected with a recombinant virus incorporating a gene encoding human chymase. (1) Chymase activity is not lost at a physiological temperature of 37 ° C. (2) completely inhibited by α-antitrypsin, chymostatin, leupeptin, aprotinin and ED
Not inhibited at all by TA. (3) shows a single band in electrophoresis after purification. 2. The recombinant human chymase according to claim 1, wherein the recombinant virus contains a gene encoding a host protein secretion signal peptide in addition to the human chymase gene. 3. The recombinant human chymase according to claim 1, wherein the recombinant virus further comprises a gene encoding an enterokinase cleavage sequence. 4. The recombinant human chymase according to any one of claims 1 to 3, wherein the host is a silkworm and the virus is a baculovirus. 5. A host or host cell is infected with a recombinant virus incorporating a gene encoding a protein secretory signal peptide of a host, a gene encoding an enterokinase cleavage sequence, and a gene encoding human chymase, and A method for producing a recombinant human chymase, which comprises obtaining an inactive recombinant human chymase from a host or a culture supernatant of a host cell after the propagation, and converting the inactivated human chymase into an active form using enterokinase. 6. The method for producing a recombinant human chymase according to claim 5, wherein the obtained recombinant human chymase has the following properties. (1) Chymase activity is not lost at a physiological temperature of 37 ° C. (2) completely inhibited by α-antitrypsin, chymostatin, leupeptin, aprotinin and ED
Not inhibited at all by TA. (3) shows a single band in electrophoresis after purification. 7. The method for producing a recombinant human chymase according to claim 5, wherein the host is a silkworm and the virus is a baculovirus. 8. A recombinant human chymase producing virus into which a gene encoding a host protein secretion signal peptide, a gene encoding an enterokinase cleavage sequence, and a gene encoding human chymase have been incorporated. 9. A method for injecting a recombinant human chymase having the following properties and a test drug into a medium for culturing cells sensitive to human chymase, and detecting a change in cells caused by the injection: A screening method for an activity that inhibits the function of human chymase. (1) Chymase activity is not lost at a physiological temperature of 37 ° C. (2) completely inhibited by α-antitrypsin, chymostatin, leupeptin, aprotinin and ED
Not inhibited at all by TA. (3) shows a single band in electrophoresis after purification. 10. A method for injecting a recombinant human chymase having the following properties and a test drug into a medium for culturing cells sensitive to human chymase, and detecting a change in cells caused by the injection: Evaluation test method. (1) Chymase activity is not lost at a physiological temperature of 37 ° C. (2) completely inhibited by α-antitrypsin, chymostatin, leupeptin, aprotinin and ED
Not inhibited at all by TA. (3) shows a single band in electrophoresis after purification.