JPH0256498A - Human calpastatin polypeptide - Google Patents

Abstract

NEW MATERIAL:A human calpastatin polypeptide expressed by the formula. EXAMPLE:A remedy for diseases caused by an overaction of a calpain, such as adult leukemia, cataract, etc. PREPARATION:For example, a cDNA prepared from a template mRNA originating from a normal human liver is linked to a vector and introduced into a host cell to form a cDNA library. A clone containing a gene coding a human calpastatin is screened from the cDNA library by using a cDNA fragment of a porcine calpastatin as a probe. The clone is then cultured and propagated and a bacteria pellet collected is treated with restriction enzymes to take up the DNA fragment, which is inserted into a vector and introduced into E. coli to transform. The resultant transformant is then cultured and a peptide expressed by the formula is obtained from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention has an amino acid sequence of human calpastatin. Concerning polypeptides and their DNA sequences.

[Conventional technology]

Calpastatin is an intracellular protein that specifically inhibits calpain, which is known as a calcium-dependent distingrothiase.

Calpastatin is widely distributed in various tissues of higher animals,
It is thought that it is mobilized when cells receive stimulation and regulates the action of activated calpain.

Calpain's roles in living organisms are known to include activation through limited decomposition of kinase enzymes such as protein kinase C1 phosphorylase kinase B, decomposition of cytoskeletal proteins, and decomposition of growth factor and hormone receptors.

Calpastatin regulates the excessive reaction of calpain by specifically inhibiting these calpain actions. Therefore, maintenance of the balance of the calpain-calpastatin system is closely related to pathological conditions.

For example, in muscular dystrophic diseases, the level of calpain increases, and it is presumed that the promotion of decomposition of myofibrils and neurofilaments by calpain is closely related to the onset of the disease.

Furthermore, it is known that calpain activity and interleukin 2 receptor activity are abnormally increased in cells infected with adult leukemia virus. This suggests that calpain acts on cytoskeletal proteins to change receptor activity, resulting in an abnormal response of cells to growth factors, etc. [Biochemistry,
Vol. 57, p. 1202, (1985)).

Furthermore, calpain is known to cleave ocular lens proteins, and its overreaction is suggested to be related to circadian syndrome. On the other hand, there is a report that calpastatin is a substance that stabilizes the steroid-receptor complex necessary for information transmission [Journal of Biolo
(Gical Chemistry) Volume 260,
2601 (1985)) and an abnormal decrease in calpastatin levels in hypertensive rats [Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications).
dBiophysicalResearch Com
municalons) Volume 145, Page 1287 (
1987) ).

As described above, calpastatin, as an endogenous protein inhibitor that regulates the action of calpain, is expected to be an effective therapeutic agent for various diseases thought to be caused by excessive action of calpain.

Furthermore, the DNA sequence encoding human calpastatin can be used for DNA diagnosis of various diseases caused by human calvine-human calpastatin imbalance.

[Problem to be solved by the invention]

Calpastatin is widely distributed in various tissues of higher animals, and has been reported to be purified from pig red blood cells and myocardium, and purified from rabbit liver. [Journal of Biochemistry, Volume 95, Page 1661 (1984), The Biochemistry Journal
nax), Volume 255, Page 97 (j986), Biochemical and Bio, Physical Research Communications, Volume 122, Page 912 (198
4) ].

Furthermore, cDNA scanning of pig and rabbit calpastatin revealed the primary structure of the amino acids. As a result, calpastatin was found to have four repeating functional units (domains 1 to 4) consisting of approximately 140 amino acid residues and a non-homologous sequence (domain L) at the N-terminus.
Domains 1 to 4 were found to have calpastatin activity on their own [FEB Letters
8 Letters), Volume 223, Page 174 (
1987)). Regarding human calpastatin, purification from red blood cells has been reported [Journal of Biochemistry Vol. 92, p. 2021 (1982) E
However, its genetic structure and amino acid sequence are still unknown. Further, an industrially advantageous manufacturing method for human calpastatin is not disclosed.An object of the present invention is to provide a human calpastatin polypeptide and a genetic engineering method for manufacturing the same.

[Means to solve the problem]

To summarize the present invention, the first F3A of the present invention relates to a polypeptide of human calpastatin, and is characterized by being represented by the following formula l. ψ C C.

L)l- Q engineering 〇－ r5 Ufu ζ− 〇J: a← Oλ J- mouth Φ a mail C” ■Ko to" L)+- CJJ: C Ij.

L++-L)CL Jif+-QI-L)L Furthermore, the second invention of the present invention relates to a base sequence encoding the polypeptide of the first invention.

Furthermore, the fifth invention of the present invention relates to a transformant into which a recombinant plasmid containing the DNA encoding the human calpastatin polypeptide of the first invention is introduced. The invention relates to a production method for culturing the transformant of the third invention and collecting the human calpastatin polypeptide of the first invention from the culture.

The present inventors selected a cDNA clone encoding human calpastatin from a human cDNA library, and determined the entire amino acid sequence of human calpastatin from its base sequence analysis. Furthermore, the cDNA was connected to an expression vector, introduced into E. coli, and the human calpastatin polypeptide was successfully expressed. The present invention was completed based on these findings.

The present invention will be explained in detail below.

A known method can be used to clone the cDNA encoding human calpastatin.

For example, polyA-containing RNA is extracted from the liver, myocardium, or human-derived cell lines, where human calpastatin is distributed, and aimed with a cellulose carrier bound to oligo-dT. Using this as a template, reverse transcriptase is applied to synthesize cDNA, and cDNA is synthesized using the Okayama-Perg method or the Gubraho7man method (Gub1θ
cD by connecting it to a plasmid or phage vector and introducing it into a host using methods such as
Create an NA library. Such libraries are also commercially available, and can be purchased, for example, from Clontic. If a portion of the cDNA has already been obtained, a cDNA library is also constructed by the primer extension method. This method is particularly effective for cloning 5' cDNAs.

In order to screen a QDNA clone encoding the desired human calpastatin from a cDNA library, it is effective to use a cDNA fragment of pig or rabbit calpastatin, the sequence of which has already been revealed, as a glove. The DNA globe can be chemically synthesized or extracted from a vector containing cDNA using restricted el#
It may be cut out and purified.

To screen a library using a DNA globe, first, the library is amplified on a plate, and the grown colonies or plaques are transferred to a nitrocellulose or nylon filter, and denatured to 5D.
Fix the NA to the filter. This filter is incubated in a solution containing a DNA globe previously labeled with 32p, etc., to form a hybrid between the DNA on the filter and the globe DNA (hereinafter, this operation is referred to as hybridization). The incubation temperature is set based on the Tm (melting temperature) of the glove used. After hybridization, nonspecific adsorption is washed away and clones that have hybridized to the globe are identified by autoradiography.

This operation is repeated to isolate clones for the next analysis.

If the recombinant is E. coli, culture it in a small amount in a test tube, extract the plasmid using a conventional method, perform a cleavage reaction with a restriction enzyme, and perform agarose or acrylamide gel electrophoresis to extract the cloned insert. Examine fragment generation. Furthermore, the electrophoretic pattern is transferred to a nitrocellulose or nylon filter, and hybridization is performed by the method described above to determine whether or not the inserted fragment forms a hybrid with the DNA probe. Finally, the base sequence of the inserted fragment is determined by a known method. When the recombinant is 77-di, clone analysis is basically performed using the same steps. A pre-cultured host E. coli was infected with cloned 7age, and 77-diDN was extracted from the lysate.
Prepare A. When the phage is λgtN, it can be grown as a lysogen and then converted into a lysate by temperature shift. It is also possible to recover DNA from infected plates. A specific method for producing phage DNA 'tA is described, for example, on page 100 of Sekisei Chemistry Experiment Lecture 1 "Gene Research Methods ■" (Tokyo Kagaku Doujin Publishing). Phage DNA is cleaved with restriction enzymes and subjected to gel permeation to confirm the inserted fragment, and homology with porcine calpastatin cDNA is determined by hybridization. Finally, the clone is confirmed by determining the base sequence.

By comparing the determined base sequence with known rabbit and pig cDNAs, the gene structure and amino acid sequence can be determined.

Furthermore, the functional units of human calpastatin can be determined by comparison with the functional units of pig and rabbit calpastatin.

In this way, the functional unit D1 of human calpastatin,
Amino acid sequences of D2, D3 and D4 and corresponding DNA
The sequence can be determined. In porcine calpastatin, each functional unit alone has inhibitory activity against calpain, but it is known that the non-homologous sequence on the N-terminal side (functional unit L) alone has no activity, and human calpastatin also has inhibitory activity against calpain. Similar results can be expected.

Therefore, all of the structural genes of the obtained cDNA,
Alternatively, a DNA sequence containing at least one functional unit among functional units 1 to 4 is connected to an expression vector so as to be expressed in an artificial host cell, and introduced into the host cell,
By culturing this, a polypeptide having human calpastatin activity can be produced. Various existing vectors can be used as the expression vector.

Calpastatin from pigs and rabbits has S-
Because it does not contain S bonds and does not have sugar chains,
Human calpastatin polypeptide, which has already been confirmed to be expressed in E. coli, can be similarly expressed in E. coli.

Confirmation of expression can be performed by determining the inhibitory activity against calpain.For example, an E. coli cell extract is added to a calpain measurement system using casein as a substrate, and the decrease in the acid-soluble fraction is measured by spectroscopic analysis. This can be confirmed by specific measurements.

Purification of human calpastatin polypeptide from E. coli uses conventional chromatographic techniques.

That is, the cultured bacterial cells are treated with lysozyme and then treated with ultrasound to obtain a supernatant, which is further heat-treated by utilizing the thermal stability of calpastatin to obtain a soluble fraction. Then, the desired peptide can be obtained by chromatography such as ion exchange or gel filtration.

As described above, according to the present invention, the primary structure and functional unit of human calpastatin polypeptide have been clarified, and it has become possible to provide a method for producing the same using genetic engineering.

〔Example〕

Examples of the present invention will be shown next, but these are not intended to limit the invention.

Example 1 Cloning of human calpastatin cDNA (1-1) Screening from QDNA library Identification and isolation of positive clones> The aDNA library was obtained from Clontic (USA). This is polyA mRNA derived from the liver of a normal person.
cDNA produced using A as a template (code number HL1
001B) was cloned into the λgtll vector. Using Escherichia coli Y 1090 strain (all bacteria used below are Escherichia! bacteria) as a host bacterium, 10 to 14 c
10,000~20 per 10 Tn square petri dishes
,000 plaques were formed. That is Y 10
90 strains were cultured overnight at 57° C. in L medium to which ampicillin and maltose were added to give a final yield of 50 μ2/g and [L4f/− (hereinafter abbreviated as L + Ap + Mal medium). This was used as a host bacteria, and 12d and 77-di solution α1-1 of this and soft agar (agarose was added to the L medium to a final concentration of 0.6%, treated in an autoclave, and kept at 50°C) ) qtst was added and spread on L-grate (hereinafter this operation will be abbreviated as brating).

This plate was then incubated at 42°C for 1 hour and then at 57°C for 8 hours. The formed 7-age plaque was transferred to a nylon membrane manufactured by Amajam (code number Hybond-N), and this was coated with CL 5 M.
NaOH, 5 min (denaturation) on a paper soaked in a solution of 1,5M NaCt, [L5M)! jsu (Trio) H
After treatment for 5 min (neutralization) on a paper soaked in a solution of 1.5 M NaC2, 2 x 5
ac (NaC117,55, sodium citrate II
L82f (with H2O2 in it)] and dried on a slip paper (hereinafter this operation will be abbreviated as filtering). U
The filter was irradiated with a v lamp for 5 minutes to fix the DNA. As a probe, a 1 kilobase Pat I fragment spanning from part of domain 1 to domain 5 of calpastatin cDNA derived from pig heart was used. That is, plasmid pc containing porcine calpastatin cDNA,
Pat sFL 715 (obtained from Kyoto University Chirus Laboratory)
Nucleogen D after l digestion
The desired fragment was obtained by separation and purification using an EAE 4000 column. The obtained fragment 140n1 was labeled with 32p using the Multiprime labeling system manufactured by Amajam (catalog code number RPN, +600Y) to obtain a probe with a specific activity of 5 X + O'cpm/nj'. 6XSSC using a panoramic view of this globe and the filter adjusted above.

1% SD8100μf/- salmon sperm DNA, 5X Dθnhardt (bovine serum albumin, polyvinylpyrrolidone, Ficoll each at α1%)
Hybridization was carried out overnight at 65° C. in approximately 55 ml of a solution containing (at a concentration of) Then 2x at room temperature
Filters were washed for 10 minutes in SSc, α1% 8DS solution. After changing the solution to a fresh one and repeating this operation three more times, the filter was washed for 60 minutes in 1X5SC, (L1% SDB) at 50°C. After removing excess water by placing the filter on Kimwipe, The Whatman 5 MM cover was removed, an intensifying screen was applied, and autoradiography was performed overnight at -70°C.As a result of autoradiography, a total of 51 positive signals were obtained.

Place the plaques at the positions corresponding to these signals on the agar and add 1-8M solution of NaCl (3.8 f.

MgSO4 7H20 2r, IM) Lis HC2pH7
, 5,50-12% gelatin 5- is dissolved in 80 ml of water, the total volume is 1 t), collected, suspended, diluted appropriately and made into a grating (approximately 300 plaques/φ93 round shear) Same as above. (hereinafter referred to as "secondary screening").

As a result, single plaques could be isolated for 10 clones.

Preparation of crude phage solution> Next, about 10' of the 7 cloned phages were plated in one round Petri dish, and incubated at 42°C for 1 hour for 37 hours.
After incubation for 6 hours at <RTIgt;C,</RTI> an 8M solution of 5- was added and gently shaken for 2 hours at 4 <0>C to elute the phages. The eluate was collected, stirred with a few drops of chloroform, and stored at 4°C (hereinafter abbreviated as plate lysate method). Approximately 5x10t using the above method.

A phage solution of PFU/- was obtained.

Isolation of lysogenic bacteria> Next, add 50 μt of the obtained 7Age solution, 30 μt of a 105-fold dilution of Y1089 strain cultured with shaking overnight in L + Ap + Mal medium, and 20 μt of an 8M solution, and stir. and incubated at 37°C for 15 minutes. By the above method, 7 out of 10 clones were able to obtain Bacillus melanogaster. Next, lysogenic bacteria were added to L+ with MflCl to a final concentration of 10 mM.
The cells were planted in Ap medium and cultured at 50°C overnight.

Preparation of λ-DNA> This late culture (L2-) was planted in L4-Ap medium supplemented with 10- of the above MgCt2, shaken and cultured,
D, When 600 nm reached α5 (3 hours after inoculation), the cells were shaken at 45°C for 15 minutes (heat induction) and then continued to shake at 37°C for 2.5 hours. 6,00
The cells were collected by centrifugation at 0 rpm for 10 minutes, and the cell pellets were suspended in an 8M solution of 12-, followed by addition of 12-chloroform and stirring to cause dissolution. After leaving it for 50 minutes,
Centrifuge at 6,000 rpm for 10 minutes to remove DNA from the upper groove.
final concentration of se and RNase each at 5μ/-150μ
m7.1 and incubated at 37°C for 10 minutes. Perform phenol extraction, phenol-chloroform extraction, and chloroform extraction, and add I/+oi- to the supernatant.
5M sodium acetate solution and twice the volume of ethanol were added to precipitate the DNA (hereinafter abbreviated as ethanol precipitation).
. After standing overnight at -20°C, centrifugation was performed at 10,000 rpm for 10 min, and the precipitate was rinsed with 80% ethanol.
0mM Tris HCl pH7.5, (
Dissolved in L 1 mM ED'I'A).

Identification of inserted fragment> 1 μt of the above DNA solution prepared, 110 times concentrated EcoR1
3 μl of buffer solution (composition is listed in the Zenshu Brewing and Genetic Engineering Reagent Catalog, hereinafter abbreviated as 10x buffer solution),
Add 50 units of EcoRl and water to make a total volume of 30 μt.
After stirring and incubating at 37°C for 2 hours, RNase was added to a final concentration of 50 At/d and further incubated for 10 minutes. Take 10 μt from the above reaction solution and 2 μt of 6-fold concentrated electrophoresis buffer (0.25% bromophenol blue (125 thixylene cyanol, 30% glycerol, hereinafter referred to as 6
x EP buffer) was added and electrophoresed on a 1.2% agarose gel, and for all 7 clones, 1
．． An 8 kb insert was identified.

<Restriction enzyme mapping of insert fragment> One clone was arbitrarily selected from among the seven clones in which insert fragments of 1.8 kb were identified and named λc819. 40 μt of DNA solution prepared from λcB19 and 10
× Buffer solution 40 μt% EcoR1200 unit and water were added to bring the total volume to 400 μt, stirred, and stirred at 57C for 2.
After incubation for 5 hours, RNA (je) was added to a final concentration of 50 At/- and incubated for an additional 10 minutes.
Incubated at ℃. After removing IGm from the above reaction system and confirming that the inserted fragment had been excised by agarose gel electrophoresis, 5M ammonium acetate was added to a final concentration of 2MK, and after stirring, ethanol precipitation was performed.

- 10 at 10,00 orpm after incubating overnight at 20°C.
Centrifuge for min, rinse the precipitate with 80% ethanol, dry the precipitate at room temperature, and add 40 μt of T.

E, dissolved in buffer. KIOμt 6xEP buffer was added to this, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the gel was stained with a 1 μf/− ethidium bromide solution for 10 minutes, and then a portion containing the desired inserted fragment was cut out from the gel under ultraviolet irradiation. This was mixed with electroextraction buffer (0,004M-) lisacetic acid, 11mM gDT.
A, pH 8, hereinafter abbreviated as BE buffer) was inserted into a dialysis tube filled with the same solution as the external solution, and the voltage was set at 17V.
/cm for 20 minutes. After gently pulling out the gel from the dialysis tube, return the tube to its original position.
A reverse current was applied for 5 seconds to elute the DNA into the buffer in the tube. Take out this DNA solution, add an equal amount of phenol/chloroform mixture and stir.6.
After centrifugation at 00 rpm for 5 minutes, the supernatant was collected (hereinafter referred to as phenol/chloroform extraction).

The supernatant DNA was precipitated with ethanol, rinsed with 80% ethanol, and then dissolved in 80 μt of TE. The obtained λ
15 μt of a DNA solution containing the insert fragment of cB19 (1.8 kl) and pUC118 (Zen Shuzo) digested with EcoR1 α2 μF, 10x ligation buffer (α5M
) Squirrel Hct (1)H7,5), CL + M Mg
C1, (11M dithiothreitol, 10mM spermidine, 2mM ATP) 2.8 units of 1T4 DNA ligase and water were added to a total volume of 25μt, stirred, and incubated overnight at 15°C.

Using 5 μt of this ligation reaction solution, JM109
The strain was transformed with x-Ga1 (5-promo 4-chloro-
3-indolyl-β-D-galactoside) and IPTG
(Isopropyl-β-D-thiogalactoside) at a concentration of 40μf/d and H'μf/d, respectively.
A recombinant with a 1.8 kl fragment inserted was obtained by plating it on a medium plate and picking white colonies. Four clones were arbitrarily selected from among the obtained recombinants, cultured overnight in 5-L-Ap medium, and then 1.5-
alkaline lysis method from the culture solution [Reference: Molecular Cloning]
Page 568, Cold Spring Harbor Laboratory (
Plasmid DNA was prepared by
μt 'I', K, dissolved in buffer.

The DNA prepared from the above four clones was converted into pUo.
11 Digest with a restriction enzyme alone whose recognition sequence is within the polylinker sequence fence of B, and then digest with the above enzyme and B.
It was digested in combination with amHl and the cleavage pattern was analyzed by electrophoresis. Figure 1 shows a restriction enzyme map. For DNA digestion, add 1 μt of the DNA solution prepared above and 10 μt of each enzyme.
Add 1 μt of twice-concentrated buffer solution (described in the Takara Shuzo Genetic Engineering Reagent Catalog, hereinafter abbreviated as 10× buffer) and 5 units of each enzyme and water to make a total volume of 10 μt, stir,
This was done by incubating at 7°C for 2 hours.

After the reaction was completed, 2 μt of SXEP buffer was added to the reaction solution, and electrophoresis was performed using a 1.2% agarose gel.

<Base sequencing of insert fragment> λ prepared previously. s19 insert fragment DNA solution 1511
gco the RF DNA of 1 and M15mp+9 (Zen Shuzo)
Add R1 digested product Q, 2 µt, 2.5 µA of 110 times concentrated ligation buffer, 2.8 units of T4 DNA ligase, and water, stir to make a total volume of 25 µt, and stir for 15 minutes.
Incubated overnight at °C.

Using 1 μt of this ligation reaction solution, JM109 strain was transformed in the same manner as described above, and X-Ga1 and IPTG
1■ each, (JM1 as L258rng indicator bacteria)
09 strain cultured overnight in L medium α2-1 soft agar (
Agar was added to B medium to a final concentration of α6%, autoclaved and heated to 50°C) 4 w&t was added, and B medium (1 ton of Bacto Tori 10 f, NaC) was added.
1Bt/L.

Recombinants containing the t a kb fragment were obtained by plating on 1% vitamin Bl) plates and selecting white plaques.

The obtained 6 recombinant 12 clones, 77-di, were extracted from plaques and an overnight culture of p5- strain JM109 was cultured in L-medium for 1 hour.
The cells were infected with a diluted solution of 7100, and cultured with shaking for 6 hours. Similar to the plasmid DNA, RF DNA of recombinant 7age was prepared by the alkaline lysis method described above and dissolved in 15 μt of TE buffer. This DNA solution 5
10x buffer for μt and HinallI 11Jt, H
lncl[[Mix 5 units with water to make a total of 10 At
After stirring, the mixture was incubated at 57°C for 2 hours. Add 2At 6xKP buffer, perform electrophoresis on t2% agarose gel, and remove 1.8 kl from the cutting pattern)
The orientation of the insert was determined (named MC82 in one orientation and MC85 in the other orientation). MC82
and MC85, two clones (MC822 and MC8
29) was produced.

MC822 and MC829ORF from the culture medium of 2o-
DNA was prepared similarly and 10μ2150μt T
A DNA solution of i1e was obtained. RFDNA of MC822
1μF (5μt) and 2μt of 10x buffer 150μt
/ml RNase solution 1 ttL%Xhol 5 units, add 5a115 units and water to make total i'20
After stirring, the mixture was incubated at 57° C. for 1 hour. 10 molecules of the reaction solution were kept at 165°C, the restriction enzyme was inactivated, 2 μt was taken out, 10 μt of 10x ligation buffer, T4 DNA ligase and water were added to make a total volume of 100 μt, and after stirring, 16
Incubated for 3 hours at ℃. The JM109 strain was transformed using 40 μt of the reaction solution in the same manner as described above, and the clones with white plaques were analyzed, and a derivative (M
C841) was obtained.

Similarly, Sacl and Accl, respectively.

Hl nam, Pet, l, BgIII + B
By using amHl and XbaI, a derivative in which the N-terminus was deleted from the site of the corresponding insertion fragment was obtained.

However, in the case of Accl, since the recognition sequence was not uniquely determined, ligation was performed after the sticky ends were made blunt ends. Similarly, starting from MC829, a derivative lacking the C-terminal side of the above-mentioned site was obtained.

In addition, the (14 kb H
lndl [fragment is ゛, M13mp18 Hlndl [[
subcloned to the site.

Using the above derivative clone, the entire nucleotide sequence of the t8kb insert was determined by the dideoxy method. The area covered by this inserted fragment on the physical map is shown in FIG. 1 as λcB19.

The determined base sequence was converted into the aDN of porcine calpastatin.
By comparison with A, the L8kb insert fragment is
It was revealed that it covers 90% of the coating region of the calpastatin gene.

(1-2) cDNA cloning by primer extension method Next, in order to obtain a clone that covers a part of functional unit 1 that is not covered by the above tskb fragment, mR
cDNA cloning was performed using the NA primer extension method.

(Identification and Isolation of Positive Clones) The mRNA derived from a normal clone was obtained from the aforementioned Clontic Co., Ltd. (code number 6510).

The primer is 5 at position 602 to 586 of the base sequence.
A 17 bp synthetic NA existing in the '→3' direction was used. mRNA 2 μm and primer (11 μV
and synthesize cDNA using AmaJam's cDNA synthesis kit. Next, the same product made by Amajam Co., Ltd.
Using the cDNA cloning system λgt10, aDNA was integrated into the EcoR1 site of 1g1;10 in a cell-free system and packaged into lambda 7age.

However, for the package, GIGAPACK Gold (GIGAPACK COLD) manufactured by Strategene was used. The host bacteria were plated using 0600Hfj strain recommended by Strategene, and placed in 10 square petri dishes.
96,000 phage were formed per plate, and this was transferred to a filter and filtered.

As a probe, an EcoRl-Xhol fragment corresponding to positions 516 to 690 of the base sequence was used.

56 μl of the aforementioned pcs8, +ox buffer, EcoR
1100: x-nit, Xhol 100 units and water were added to make 400 μt, stirred, and incubated at 57° C. for 2 hours. After the reaction is completed, 1.
After electrophoresis was performed on 2 layers of agarose gel and a portion containing the desired fragment was excised, the DNA was extracted electrically as described above to obtain 2.2 μ2. Of these, 01μ2 is
#labeling with 3Zp using the multi-prime labeling system described above yielded a glove with a specific activity of 2XIG'CPM/μt.

Hybridization was performed using this probe 3X+0'CPM and the filter prepared previously. The filters were then washed and autoradiographed. Since one positive signal was obtained, we performed a secondary screening and isolated a single Gzik (λas 151
).

(Preparation of DNA of λ, 151) Next, a phage was prepared from a 40-ml culture solution by a liquid culture method (Gene Research Method, p. 100, Tokyo Kagaku Doujin Publishing), and 24 μ2 of DNA was obtained from it.

(Identification of inserted fragment) Previously obtained DNA fragment 5 and 10x buffer 5bL1gc
oR150 units were added to give a total volume of 30βt, and after stirring, the mixture was incubated at 57°C for 2 hours. The reaction solution was analyzed on a 1.2% agarose gel, and the inserted fragment of (L 5 kl) was identified.

(Determination of base sequence of inserted fragment) 20 μf of the previously obtained phage DNA and 60 μL% EcoR1100 units of 10× buffer were added to give a total volume of 600 μt, stirred, and reacted at 57C for 2 hours. The reaction solution was concentrated by ethanol precipitation, and the DNA
was dissolved in 40 μt of TE. This was subjected to 1.2% 7-garose gel electrophoresis, a portion containing the desired inserted fragment was excised, and the DNA was electrically extracted to obtain 100 nf. The obtained DNA fragment 50 rsf and RFM13
Add 50nf of ECORI digest of mp+9, 1μt of jox ligation buffer, 2.8 units of T4 DNA ligase and water to make 10μt, stir,
Partially incubated at ℃.

JM109 strain was transformed using 1 μt of the ligation reaction solution, and a recombinant clone was obtained in the same manner as described above. Using this clone, 1
The nucleotide sequence of the inserted fragment (5 kl) was determined. The area covered by this inserted fragment on the physical map is shown in FIG. 1 as λca131. Examples (1-1) and (1-2
) revealed that clones covering part of the functional units of human calpastatin and functional units 1 to 4t were obtained.

(+-3) cDNA cloning of the N-terminal region In order to obtain a cDNA clone covering the N-terminal region, the N-terminal fragment of the existing cDNA clone was extracted from a cDNA library created using random primers. Screening was conducted using gloves. the result,
A clone covering up to 90 base pairs upstream of the start codon of calpastatin protein was obtained. The process is shown below.

(Identification and Isolation of Positive Clones) A cDNA library was prepared in the same manner as shown in (1-2) using the aforementioned mRNA 5μ2 and random hexamer 1.5μ2 as a primer. Next, as a probe, the aforementioned EcoRl-Acel of λc819 corresponding to positions 590 to 465 of the base sequence was used.
Using this fragment, approximately 1 clone having a fragment homologous to this fragment is extracted.
Screening was performed from 00,000 recombinants. the result,
Four positive clones were isolated.

(Analysis of insert fragment) Next, the insert fragment was identified in the same manner as shown in (i-2), and the base sequence was analyzed. As a result, one clone (λc,
143) was found to cover the region from -96 to 1294, which includes the start codon of calpastatin protein. The area covered by λcs145 on a physical map is shown in Figure 1. From the results of the nucleotide sequence analysis in Examples (1-1), (1-2), and (1-5), the entire nucleotide sequence of the human calpastatin aDNA and the entire amino acid sequence of the protein of the present invention are revealed. It became K.

Example 2 Construction of an expression plasmid (2-1) Construction of a plasmid expressing human calpastatin λc819 and λ were constructed using the following procedure. 8131 insertion Ec
The oRI fragments are spliced together at the Accl site in the area where the sea fences overlap, and this is connected to PUC1.
An expression plasmid (named pH08121) inserted in frame into the Neo I site of 19(N) was constructed.

After digesting pcsa 60 at with gcoRl and ethanol precipitation, 10x buffer 45 At% AcC12
70 units and water were added to make a total volume of 450 μt, stirred, and incubated at 57°C for 2.5 minutes.
The reaction was terminated by adding 7Qltt (Dα2 MEDTA) and partially digested with plasmid DNA At-Accl. Next, electrophoresis was performed on a 1% agarose gel.
The part containing the EcoRl-Ace 1 partially digested fragment of kb was excised and the DNA was electrically extracted (actually, 4μ was obtained by mixing it with the undigested gcoR1 fragment).
. 2 μt of this partially digested fragment and 0.0 μt of the BamHl-Accl fragment excised and prepared from λas 151. ．． 511 pUC119 (N) was double digested with EcoRl and BamHl α2/Jf.

2 µt of 10X ligation buffer and water were added to make a total volume of 20 µt, stirred, and reacted overnight at 15°C.

After keeping the reaction solution at 70°C for 10 minutes to inactivate the ligase, take out 10 μt and add 10×TA buffer S A to this.
Add t, amal 10 units, and water to make a total amount of 50
μt and incubated at 57°C for 1 hour. Of these, 15 μt was used to transform strain 7M109, and p
Hcs+5 was obtained.

1) Prepare HC81 plasmid DNA, α8μf
After digesting the oDNA with BamHl, phenol extraction and ethanol precipitation were performed to recover the DNA. Then 10x
Buffer (70mM) Lis HC2゜pH7,5,200m
1.5 μt of M NaC1, 70mM MgC1*), 2 units of Flenow enzyme, and dNTP mixture (1 each
mM) and water were added to make a total volume of 15 μt, stirred, and incubated at 37° C. for 10 minutes.

Next, add 12 mar of Ncal linker [d(p
CAGCCATGGCTH) ) (L 1 μ?
, 200mMdTTl ttlll 0mMA'
Add l' P α5At, T4 DNA ligase "L8 unit" and water, stir to make a total volume of 20 μt, and add 1
Partial ink evacuation was performed at 5°C.

After removing the excess linker with a Sepharose CL-4B column (Pharmacia, 115 M NaCl, equilibrated with lithium hydrochloric acid (pH 7.5), α1 mM EDTA) containing the compound mts-, the MgCl snow was terminated. Concentration t O
Add 100 units of Ncol to make a total volume of 550 μt, and incubate at 57° C. for 25 hours. Next, keep it at 70°C for 10 minutes and
After inactivation, 250μ was taken out from the reaction system and added to the final concentration of A'I'P and D'l"rtl (12 m
M was added to 10 mM, and 15 units of T4 DNA ligase was added, followed by incubation at 15°C overnight. Next, the reaction solution was kept at 70°C for 10 minutes to inactivate the ligase, and then 5M NaC15At, 5all
After adding 5Q units and incubating for 2 hours at 37°C, the mixture was concentrated using a concentrator and dialyzed against water (final volume 63 μt). 25 bL from now on
was extracted and transformed into 7M109 strain, pHcs1
I got 21. Figure 2 shows the construction process as a process diagram.

E. coli introduced with pHc8121, 7M10? 138
cherichia coll JMl 09 /
It was expressed as pHc8+21 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.
MP-9989)).

Next, downstream of the structural gene of calpastatin at pHcs 121, the pI Nm-ompA vector [K, Nakamura and M+ Inoue (K+Nakamura, M, Inoue) was inserted.
uye), Cell (Ce1l) Volume 18, No. 1109
(1979)) was introduced by the following procedure.

ptJc 118(N) was cut with Neol and Sall, and this vector fragment 10n2 and pINl[-om
50 nlF of the EcoRl-8all fragment containing the pA terminator and 50n2 of the Nco 1-EcoR1 fragment of the calpastatin structural gene at pHcs 121 were mixed, 'r4 DNA ligase was added, and the mixture was reacted overnight at 15°C. Using 3 μt of this 15 μt of reaction solution, J
Transform the u109 strain and adjust the pHc as shown in Figure 2.
I got s131.

Next, follow the steps below to determine λ. The inserted EcoR1 fragment of 8143 and pHcs 151 were joined together at the restriction enzyme site located in the region where the sea fence overlapped, and the Nco
An expression plasmid pHc81 B + was constructed by ligating them together so that they fit in frame at the l site.

λ. gcoR1 fragment 5 excised and prepared from 5145
0nf, 10nf obtained by digesting pUc119 (N) with EQORI was reacted at 15°C for 30 minutes using a TakaRa ligation kit. This reaction solution 60
JM using 20 μt from tit? 09 strain was transformed to obtain pHc851. The H1nfl fragment excised from pHcs51 and prepared was subjected to end repair with Flenow 7 fragment, and then digested with Bcnl to create blunt-end-B
An l fragment was obtained. In addition, the Ncol-Xnol fragment prepared by cutting out from pHcs 121 was digested with BcnI to obtain a Banl-Xhol fragment. Furthermore, pHc
After s + 21 was digested with Neo I, end repair was performed using the Flenow fragment, and it was digested with Xhol. 1
% low melting point agarose gel, 4.5kl
), cut out the portion containing the blunt end-Xhol fragment, and
DNA was collected. The five fragments listed above, blunt ends -
Ban l fragment 50 nf, Ban l -Xho l
Fragment 50nf, blunt end - Xho 1 fragment 50nf
was reacted at 15° C. for 50 minutes using a Takara ligation kit. 60μt to 20μt of this reaction solution
Transform JM109 strain using pHcs 181
I got it. Figure 2 shows the construction process as a process diagram.

Escherichia coli JM109 introduced with pHcs 181 was
herichia coli JM 109/pHcs
+ 81 and deposited with the Institute of Microbiology, Agency of Industrial Science and Technology
-10"18B)].

Example 3 Expression of human calpastatin polypeptide in Escherichia coli obtained in Example 2
J Ml 09/pHcs 181 was planted in 5-L medium and cultured with shaking at 37°C. When the medium of logarithmic growth was reached, IP'rG was added to a final concentration of 2 mM, and the culture was continued for an additional 4 hours. went. After culturing, the bacterial cells were cooled to 0°C, and NaNg was added to the final concentration (LO2%).
After stirring, bacteria were collected. Discard the supernatant and remove the bacterial cells (L6-
M buffer (20mM) of Lis HCt (pH 7,5),
+mM gDTA, +mM EGTA
.

Q, 2mM PMSF, 5mM 2-mercagtoethanol] and centrifuged, CL5g
Suspend lysozyme in M buffer at a final concentration of cL5m9.
/- and incubate at D°C for 10 minutes. Next, freeze-thaw (-70°C #20°C) was repeated 5 times, centrifuged at 14,000 rpm for 20 minutes, and the supernatant was
℃ for 10 minutes. Further this at 14,000 rpm
The mixture was centrifuged for 10 minutes and the upper groove was collected. The calpastatin activity of the thus obtained preparation was measured by observing the inhibition of calpain as shown below (Journal Op Biochemistry, Vol. 95, p. 1759 (1984)). The sample, calpain, 40 μt of 2-ticasein, 20 μt of 6/fRt cysteine solution, and water were mixed to give a total volume of 180 μt.

Next, after preheating to 30°C for 5 minutes, 20μ of 1.50 mM calcium chloride was added, mixed, and incubated at 50°C for 50 minutes. Next, the reaction was stopped by adding 200μ of 5% TCA, centrifuged at 5.500 rpm for 10 minutes, and the supernatant was collected.

Of these, α2- was removed and monoiodoacetic acid solution ([L 7
M NaCO3/α1N NaOH and 2N monoiodoacetic acid/2N NaOH mixed at 1=1) 1
00μt, and then Lowry reagent (α5%CuSO4
and [11% potassium tartrate, 2% Na1CO@/α1N
1- (a mixture of NaOH in a ratio of 1:1:50) was added, mixed, and allowed to stand for 10 minutes.

Next, add 100μt of 1N phenol reagent, stir immediately, leave for 50 minutes or more, and then add A. 5 Onm was measured, and the carbine inhibitory activity was calculated from this.

10 μt of the prepared solution obtained by the above method inhibited porcine erythrocyte calvine I by 66% in the (L2m calpain reaction system). Next, the active substance polypeptide was purified, and its structure included the structure shown by D5 above. It was confirmed that it was a polypeptide.Escherichia coli JMI09, which does not retain Grasmid, was treated in the same manner, but no inhibitory activity was observed.

〔Effect of the invention〕

From the above results, the present invention has revealed the entire amino acid sequence of human calpastatin and its DNA sequence, and provided a genetic engineering method for producing human calpastatin polypeptide.

[Brief explanation of the drawing]

Figure 1 shows the restriction enzyme map and functional unit structure of human calpastatin cDNA, and Figure 2 shows the construction process of recombinant grasmid to express a polypeptide having the entire amino acid sequence of human calpastatin. This is a process diagram.

Claims (1)

[Claims] 1. A human carvastatin polypeptide represented by the following formula I. [There is a gene sequence]... [I] [There is a gene sequence] 2. A base sequence encoding the polypeptide according to claim 1. 3. A transformant into which a recombinant plasmid containing the DNA encoding the human carvastatin polypeptide according to claim 1 has been introduced. 4. A method for producing human carvastatin polypeptide, which comprises culturing the transformant according to claim 3 and collecting the human carvastatin polypeptide according to claim 1 from the culture.