JP2657252B2 - Human calpastatin-like polypeptide - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a polypeptide having an amino acid sequence of human calpastatin and a DNA sequence thereof.

[Conventional technology]

Calpastatin is an intracellular protein that specifically inhibits calpain, known as a calcium-dependent cysteine protease. Calpastatin is widely distributed in various tissues of higher animals, and is thought to regulate the action of activated calpain by being recruited by stimulating cells. As the role of calpain in the living body, activation by the limited degradation of kinase enzymes such as protein kinase C and phosphorylase kinase B, degradation of cytoskeletal proteins, degradation of growth factor and hormone receptors, and the like are known.

Calpastatin regulates calpain overreaction by specifically inhibiting the action of these calpains. Therefore, maintaining the calpain-calpastatin system balance is closely related to the disease state.

For example, in muscular dystrophy disease, the level of calpain is high, and the degradation of myofibrils and neurofilament is promoted by calpain.
It is presumed to be closely related to the onset.

In addition, 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 alter receptor activity, resulting in an abnormal response of cells to growth factors and the like, leading to pathogenesis (Biochemistry, Vol. 57, No. 1202). Pp. (1985)]. Furthermore, calpain is known to cleave ocular lens proteins, suggesting that its overreaction is associated with cataract. On the other hand, it has been reported that the steroid-receptor complex stabilizing substance required for signal transmission is calpastatin [Journal of Biological Chemistry (Journal).
of Biological Chemistry) Volume 260, Page 2601 (198
5)] and an abnormal decrease in calpastatin levels in hypertensive rats [Biochemical and Biophysical Research Communicats].
ions) Vol. 145, p. 1287 (1987)].

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

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

[Problems to be solved by the invention]

Calpastatin is widely distributed in various tissues of higher animals,
Purification from porcine erythrocytes and myocardium and purification from rabbit liver have been reported. [Journal of Biochmistry, Vol. 95, p. 1661 (19
84), The Biochemical Journal
Journal), Vol. 235, p. 97 (1986), Biochemical and Biophysical Research Comi-Unices, Vol. 122, p. 912 (1984)].

In addition, cDNA cloning of pig and rabbit calpastatin revealed the primary structure of the amino acids. As a result, calpastatin is composed of about 140 amino acid residues.
It was found that the two repeating functional units (domains 1 to 4) and the N-terminal had a non-homologous sequence (domain L), and it was found that domains 1 to 4 had calpastatin activity alone in each domain [ FEBS Lette
rs), Volume 223, Page 174 (1987)]. Purification of human calpastatin from erythrocytes has been reported [Journal of Biochemistry, Vol. 92, No. 2021.
(1982)], but the gene structure and amino acid sequence are still unknown. Also, there is no disclosure of an industrially advantageous method for producing human calpastatin.

An object of the present invention is to provide a human calpastatin-like polypeptide and a method for producing the same by genetic engineering.

[Means for solving the problem]

To summarize the present invention, the first invention of the present invention is a functional unit of human calpastatin and has the following formula D1, D2, D3 or D4
In the polypeptide, or the following formula D1, D2, D3,
D4 relates to a human calpastatin-like polypeptide bound in this order.

Further, the second invention of the present invention relates to a nucleic acid encoding the polypeptide of the first invention.

Further, the third invention of the present invention relates to a transformant into which a recombinant plasmid containing the nucleic acid of the second invention of the present invention has been introduced, and the fourth invention of the present invention relates to the transformant of the third invention. The present invention relates to a production method for culturing a transformant and collecting the human calpastatin-like polypeptide of the first invention of the present invention from the culture.

The present inventors selected a cDNA clone encoding human calpastatin from a human cDNA library, and analyzed its base sequence from the functional units D1, D2, D3 of human calpastatin.
And the amino acid sequence of D4 was determined. Furthermore, the cDNA was connected to an expression vector, introduced into E. coli, and the human calpastatin-like polypeptide was successfully expressed. The present invention has been completed based on these findings.

 Hereinafter, the present invention will be described specifically.

Known methods are used for cloning the cDNA encoding human calpastatin. For example, RNA containing poly-A is extracted from liver or cardiac muscle where human calpastatin is distributed, or a cell line derived from human, and this is oligo-d.
Purification is performed using a cellulose carrier to which T is bound. Using this as a template, a reverse transcriptase is allowed to act, and cD
After performing NA synthesis, the plasmid was connected to a plasmid or phage vector by a method such as the Okayama-Berg method or the coupler-Hoffmann method (Gubler-Hoffmann method), and introduced into a host.
Create an NA library. Such libraries are also commercially available and can be purchased, for example, from Clonetech. When a part of cDNA has already been obtained, a cDNA library is also prepared by a primer extension method. This method is particularly effective for cloning the 5 'cDNA.

To screen a cDNA clone encoding the desired human calpastatin from a cDNA library,
It is effective to use a cDNA fragment of porcine and rabbit calpastatin whose sequence has already been determined as a probe. The DNA probe may be chemically synthesized, or may be cut out of a vector containing cDNA with a restriction enzyme and purified.

As a means of screening the library with a DNA probe, first, the library is amplified on a plate, and the grown colonies or plaques are transferred to a nitrocellulose or nylon filter, and DNase is denatured.
Fix A to the filter. This filter is incubated in a solution containing a DNA probe previously labeled with ³² P or the like to form a hybrid between the DNA on the filter and the probe DNA (hereinafter, this operation is referred to as hybridization). The temperature of the incubation is set using the Tm (melting temperature) of the probe used as a guide.
After hybridization, wash off non-specific adsorption,
By autoradiography, a clone that has formed a hybrid with the probe is identified. This operation is performed again to isolate the clone, and the next analysis is performed.

When the recombinant is Escherichia coli, a small amount of culture is performed in a test tube or the like, the plasmid is extracted by a conventional method, a cleavage reaction is performed with a restriction enzyme, and the plasmid is cloned by agarose or acrylamide gel electrophoresis. Check the production of inserted fragments. Further, the electrophoresis pattern is transferred to a nitrocellulose or nylon filter, and hybridization is performed by the above-described method to examine 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 a phage, the clone is analyzed in basically the same steps. Infect the cloned phage into host Escherichia coli that has been cultured beforehand, and extract phage DNA
Is prepared. When the phage is λgt11, it can be grown as a lysogen and then lysed by temperature shift. It is also possible to recover DNA from infected plates. The specific method for preparing phage DNA is described, for example, in Seijisei Kagaku Jikken Koza 1 “Genetic Research Method II”, page 100 (Tokyo Kagaku Dojin Publishing). The phage DNA is cleaved with a restriction enzyme and subjected to gel electrophoresis to confirm the inserted fragment. Further, the homology with porcine calpastatin cDNA is examined by hybridization. Finally, the clone is confirmed by determining the nucleotide sequence.

By comparing the determined nucleotide sequence with known rabbit and porcine cDNAs, the gene structure and amino acid sequence thereof can be known.

Furthermore, the functional unit of human calpastatin can be known from comparison with the functional unit of pig and rabbit calpastatin.

Thus, the functional unit D1 of human calpastatin,
The amino acid sequence of D2, D3 and D4 and the corresponding DNA sequence can be determined. In porcine calpastatin,
It is known that each functional unit alone has an inhibitory activity against calpain, but it is known that the N-terminal heterologous sequence (functional unit L) alone has no activity, and similar results can be expected with human calpastatin. . Therefore, it was obtained
All of the cDNA structural genes or a DNA sequence containing at least one of the functional units 1 to 4 is connected to an expression vector so that the DNA sequence can be expressed in an appropriate host cell, and introduced into the host cell. By culturing
A polypeptide having human calpastatin-like activity can be produced. Various existing vectors can be used as the expression vector.

Pig and rabbit human calpastatin has been confirmed to be expressed in Escherichia coli because it does not contain an SS bond in the molecule and does not have a sugar chain, and human calpastatin-like polypeptide has also been confirmed. Similarly, it can be expressed in E. coli.

Expression can be confirmed by measuring the inhibitory activity against calpain.For example, a cell extract of Escherichia coli is added to a calpain measurement system using casein as a substrate, and the decrease in the acid-soluble fraction is determined. It can be confirmed by optical measurement.

For purification of human calpastatin-like polypeptide from Escherichia coli, a usual chromatographic technique is used. That is, the cultured cells are treated with lysozyme and then subjected to ultrasonic treatment to obtain a supernatant, and further, heat treatment is performed 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 and gel filtration.

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

〔Example〕

Next, examples of the present invention will be described, but these do not limit the present invention.

Example 1 Cloning of human calpastatin cDNA (1-1) Screening from cDNA library <Identification and isolation of positive clone> The cDNA library was obtained from Clonetech (USA). In this method, cDNA (code number HL1001B) produced using polyA mRNA derived from normal human liver as a template was used as λgt11
The vector was cloned into a vector. Escherichia coli strain Y1090 (all bacteria used below are Escherichia coli) was used as a host bacterium.
00-20,000 plaques were formed. Ie Y1090
The strain was cultured overnight at 37 ° C. in an L medium supplemented with ampicillin and maltose at final concentrations of 50 μg / ml and 0.4 g / ml, respectively (hereinafter abbreviated as L + Ap + Mal medium). Using this as a host bacterium, 0.2 ml of the bacterium, 0.1 ml of phage solution, and 9 ml of soft agar (agarose was added to L medium to a final concentration of 0.6%, treated with auto-oleave, and kept at 50 ° C.) In addition, it was spread on an L-plate (hereinafter, this operation is abbreviated as plating).

Next, the plate was incubated at 42 ° C. for 1 hour and then at 37 ° C. for 8 hours. The phage plaque thus formed was transferred to a nylon membrane (code No. Hybond-N) manufactured by Amashiam, and the plaque was immersed in a 0.5 M NaOH / 1.5 M NaCl solution for 5 minutes (denatured) on a 0.5 M Tris (Tr)
is) on a piece of paper dipped in a solution of HCl (pH 7.0) 1.5M NaCl.
After the treatment (neutralization) for 2 minutes, it was rinsed in 2 × SSC [17.53 g of NaCl, 8.82 g of sodium citrate (in H ₂ O 1)], and dried on paper (hereinafter, this operation is abbreviated as filter treatment). . Irradiate this filter with a UV lamp for 5 minutes,
Was fixed. As a probe, a 1 kilobase Pst I fragment spanning from domain 1 to domain 3 of calpastatin cDNA derived from pig heart was used. That is, the plasmid pCSFL713 containing porcine calpastatin cDNA (obtained from Kyoto University Virus Research Institute) was digested with Pst I and then treated with nucleodiene (N
ucleogen) obtained by separating and purifying the target fragment using a DEAE4000 column. 140 ng of the obtained fragment was labeled with ³² P using a multiprime labeling system (catalog code No. RPN.1600Y) manufactured by Amasyam, and 5 × 10 ³ cpm /
μg of the probe having a specific activity was obtained. Using the total amount of this probe and the filter prepared above, 6 × SSC, 1% SDS10
0 μg / ml salmon sperm DNA, 5 × Denhardt
(Bovine serum albumin, polyvinylpyrrolidone, and ficoll at a concentration of 0.1% each) were contained in about 35 ml of a solution at 65 ° C. overnight for hybridization. Next, the filter was washed in a 2 × SSC, 0.1% SDS solution at room temperature for 10 minutes. The solution was replaced with a new one, and this operation was repeated three more times, followed by washing in 1 × SSC, 0.1% SDS at 50 ° C. for 60 minutes. After the filter was placed on a Kimwipe to remove excess water, a Wattman 3MM paper was attached, an intensifying screen was applied, and an autoradiograph was performed at -70 ° C overnight. As a result of performing an autoradiograph, a total of 51 positive signals were obtained.

Each agar position plaques corresponding to these signals, 1 ml of SM solution _{(NaCl5.8g, MgSO 4 · 7H 2} O in 2 g, 1M Tris HCl pH7.5,50ml, dissolve 2% gelatin 5ml of water 800ml , The total amount being 1), suspended, appropriately diluted and plated (about 300 plaques / φ9 cm round plate), and the same operation as above was performed (hereinafter abbreviated as secondary screening). As a result, single plaques could be isolated for 10 clones.

The phage which can then be cloned <Preparation of phage crude liquid> was Pureteingu 10 about ^five to one round Shiyare, 1 hour at 42 ° C., after 6 hours Inkiyubeto at 37 ° C., the SM solution of 5ml Plus 4
The phage was eluted by gentle shaking at 2 ° C. for 2 hours. The eluate was collected, stirred with a few drops of chloroform, and stored at 4 ° C. (hereinafter abbreviated as plate lysate method). A phage solution of about 3 × 10 ¹⁰ PFU / ml was obtained by the above method.

<Separation of lysogen> Next, the above-mentioned 50 μm of the phage solution and the Y1089 strain were L + A
Although shaking culture overnight at p + Mal medium 10 ⁵ fold dilutions 30
Add the μ and 20 μ SM solutions and stir at 37 ° C.
Incubated for 15 minutes. By the above method, lysogens could be obtained for 7 out of 10 clones. Next, the lysogen was seeded on an L + Ap medium supplemented with MgCl ₂ so that the final concentration was 10 mM, and cultured at 30 ° C. overnight.

<Preparation of λ-DNA> 0.2 ml of the overnight culture was added to 10 ml of the above-mentioned MgCl _2.
Inoculated in + Ap medium and cultured with shaking. When OD600nm reached 0.5 (3 hours after inoculation), shaking was performed at 45 ° C for 15 minutes (heat induction), and then shaking was continued at 37 ° C for 2.5 hours. . The cells were collected by centrifugation at 6,000 rpm for 10 minutes, the cell pellet was suspended in 0.2 ml of an SM solution, and 0.2 ml of chloroform was added to stir the cells. After leaving for 30 minutes, 6,000r
After centrifugation at pm for 10 minutes, DNase and RNase were added to the supernatant to a final concentration of 5 μg / ml and 50 μg / ml, respectively.
Incubated for minutes. Perform phenol extraction, phenol-chloroform extraction, and chloroform extraction.
A 1/10 volume of sodium acetate solution and 2 volumes of ethanol were added to precipitate DNA (hereinafter abbreviated as ethanol precipitation). After leaving at −20 ° C. overnight, the mixture was centrifuged at 10,000 rpm for 10 minutes, the precipitate was rinsed with 80% ethanol, the precipitate was dried, and 50 μl of TE buffer (10 mM Tris HCl pH7.
5, 0.1 mM EDTA).

<Identification of Insertion Fragment> 1 μm of the prepared DNA solution, 10 μl concentrated EcoRI buffer (the composition is described in the catalog of reagents for Takara Shuzo and genetic engineering, hereinafter abbreviated as 10 × buffer) 3 μ, EcoR I30 unit Add water and stir to a total volume of 30μ and stir at 37 ℃
After incubation for 2 hours in RNase, the final concentration of RNase was 50 μg / m
l and incubated for a further 10 minutes.
10 μl is taken out of the above reaction solution and 2 μl of a 6-fold concentrated electrophoresis buffer (0.25% bromophenol blue, 0.25%
% Xylene cyanol, 30% glycerol, below, 6 ×
(Abbreviated as EP buffer) and electrophoresed on a 1.2% agarose gel to identify a 1.8 kb insert for all seven clones.

<Preparation of Restriction Enzyme Map of Insertion Fragment> One clone was arbitrarily selected from seven clones in which a 1.8 kb insertion fragment was identified, and named λcs19. λcs19
DNA solution 40μ prepared from 10 × buffer 40μ, EcoR
I200 unit and water were added to stir the mixture to a total volume of 400 μm, the mixture was incubated at 37 ° C. for 2.5 hours, RNase was added to a final concentration of 50 μg / ml, and the mixture was further incubated at 37 ° C. for 10 minutes. 10 ml was taken out from the above reaction system, and after confirming that the inserted fragment was cut out by agarose gel electrophoresis, 5 M ammonium acetate was added to a final concentration of 2 M, and after stirring, ethanol precipitation was performed.

After standing at −20 ° C. overnight, centrifuged at 10,000 rpm for 10 minutes,
The precipitate was rinsed with 80% ethanol, the precipitate was dried at room temperature, and dissolved in 40 µ of TE buffer. To this was added 10 μ６ of 6 × EP buffer and electrophoresed on a 1% agarose gel. After the electrophoresis, the gel was stained with a 1 μg / ml ethidium bromide solution for 10 minutes, and a portion containing the target insert was excised from the gel under ultraviolet irradiation. This was diluted with a buffer for electric bleeding (0.004 M-Tris acetic acid, 0.1 mM EDTA, pH 8, below E
The buffer was inserted into a dialysis tube filled with E buffer, and the external solution was also filled with the same solution, and energized at 17 V / cm for 20 minutes. After the gel was gently withdrawn from the dialysis tube, the tube was returned to its original position and a reverse current was applied for 45 seconds to elute the DNA into the buffer in the tube. This DNA solution was taken out, an equal volume of a phenol / chloroform mixture was added thereto, and the mixture was stirred, centrifuged at 6,000 rpm for 5 minutes, and the supernatant was recovered (hereinafter abbreviated as phenol / chloroform extraction).

The DNA of the supernatant was precipitated with ethanol, rinsed with 80% ethanol, and then dissolved in 80 µ of TE. Of the obtained λcs19
15 μl of a DNA solution containing a 1.8 kb insert and 0.2 μg of pUC118 (Takara Shuzo) digested with EcoRI, 10 × ligation buffer (0.5 M Tris HCl (pH 7.5), 0.1 M MgCl ₂ , 0.1 M
Dithiothreitol, 10 mM spermidine, 2 mM ATP) 2.5
mu, plus the T ₄ DNA ligase 2.8 Yunitsuto and water, the total amount 25
The mixture was stirred as μ and incubated at 15 ° C. overnight.

Using 5 μg of the ligation reaction mixture, the JM109 strain was transformed and X-Gal (5-bromo-4-chloro-3-
Indolyl-β-D-galactoside) and IPTG (isopropyl-β-D-thiogalactoside) were each 40 μg / ml,
A recombinant having a 1.8 kb fragment inserted therein was obtained by selecting white colonies on a plate of an L + Ap medium containing 19 μg / ml. Four clones are arbitrarily selected from the obtained recombinants, cultured overnight in 5 ml of L-Ap medium, and then subjected to an alkaline lysis method from a 1.5 ml culture [Reference: Molecular Cloning No. 368].
Page, Cold Spring Harbor Laboratory (198
2)], and dissolved in 15 μl of TE buffer. The DNAs prepared from the above four clones were each digested with a restriction enzyme having a recognition sequence alone in the polylinker sequence of pUC118, and then digested with the above enzyme in combination with BamHI, and the cleavage pattern was determined by electrophoresis. Analyzed. FIG. 1 shows a restriction enzyme map. For digestion of DNA, 1μ of sanely prepared DNA solution, 10 times concentrated buffer for each enzyme (described in the reagent catalog for Takara Shuzo Gene Engineering,
This was performed by adding 1 μ of a 10 × buffer solution, 5 units of each enzyme and water to a total volume of 10 μ, stirring, and incubating at 37 ° C. for 2 hours. After the reaction,
The reaction solution was added with 2 μm of 6 × EP buffer, and electrophoresed using a 1.2% agarose gel.

<Determining the nucleotide sequence of the inserted fragment>
0.2 µg of Eco DNA digested from 19 (Takara Shuzo) RF DNA,
2.5 μl of 10-fold concentrated ligation buffer, T ₄ DNA ligase
2.8 units and water were added to the mixture, and the mixture was stirred at a total volume of 25 μm and incubated at 15 ° C. overnight. Using the ligation reaction solution (1 μm), the JM109 strain was transformed in the same manner as described above, and X-Gal and IPTG were used as indicator bacteria at 1 mg and 0.238 mg, respectively.
4 ml of soft agar (Agar added to B medium to a final concentration of 0.6%, treated in an autoclave, and then heated to 50 ° C)
And B medium (Bacto tryptone 10g, NaCl 8g /
1% Vitamin B ₁ ) was spread on a plate, and a white plaque was selected to obtain a recombinant having a 1.8 kb fragment inserted therein.

Phages of the obtained 12 clones of the recombinants were taken from plaques, infected with 5 ml of an overnight culture of the JM109 strain diluted 1/100 with L-medium, and cultured with shaking for 6 hours. The RF DNA of the recombinant phage was prepared in the same manner as the plasmid DNA by the alkaline lysis method described above, and dissolved in 15 µ of TE buffer. 5μ of this DNA solution and 10 × buffer 1 for Hind III
μ, Hind III5 unit and water were mixed to make a total volume of 10 μ, stirred, and incubated at 37 ° C. for 2 hours.
2 μm of 6 × EP buffer was added, electrophoresed on a 1.2% agarose gel, and the direction of the 1.8 kb insert was determined from the cleavage pattern (one direction was named MCS2 and the other direction was named MCS5). Name). Two clones (MCS22 and MCS29) in which the insert of λcs19 was inserted into the EcoRI site of M13mp19 in different directions in the same manner as MCS2 and MCS5 were prepared.
Named). From 20 ml of culture, MCS22 and MCS29
Was prepared in the same manner to obtain a DNA solution of 10 μg / 50 μTE. 1 μg (5 μ) of RF DNA of MCS22, 2 μ of a 10 × buffer, 1 μ of a 50 μg / ml RNase solution, Xho I5 unit, Sal I5 unit and water were added to make a total volume of 20 μ. After stirring, the mixture was incubated at 37 ° C. for 1 hour. After the reaction, keep at 65 ° C for 10 minutes, and inactivate the restriction
Take out 10 μl of ligation buffer 10 μ, T ₄
DNA ligase and water were added to make the total amount 100 μ, stirred, and incubated at 16 ° C. for 3 hours. 40 of reaction solution
Using μ, the lower part of JM109 was transformed in the same manner as described above, clones of white plaques were analyzed, and a derivative (MCS
41).

Similarly, Sac I, Acc I, Hind III, Ps
By using tI, BglII + BamHI, and XbaI, a derivative in which the N-terminal was deleted from the site of the corresponding inserted fragment was obtained. However, in the case of Acc I, since the recognition sequence was not uniquely determined, ligation was performed after the sticky end of the terminal was made blunt. MC in the same way
By starting from S29, a derivative was obtained in which the C-terminal of the above site was deleted. Further, the 0.4 kb HindIII fragment within the 1.8 kb fragment was subcloned into the HindIII site of M13mp18.

Using the above derivative clone, the entire nucleotide sequence of the 1.8 kb insert was determined by the dideoxy method. The region on the physical map covered by the inserted fragment is shown in FIG. 1 as λcs19. By comparing the determined nucleotide sequence with the cDNA of porcine calpastatin, the 1.8 kb insert fragment was found to be the 9th region of the coding region of the calpastatin gene.
It has become clear that it will cover the cost.

(1-2) cDNA Cloning by Primer Extension Method Next, in order to obtain a clone covering a part of the functional unit 1, which was not covered by the above 1.8 kb fragment, cDNA cloning by mRNA primer extension method was performed.

(Identification and Isolation of Positive Clones) mRNA derived from normal human liver was obtained from the aforementioned Clonetech (code No. 6510). The primer exists at a position from 524 to 508 in the base sequence in a 5 ′ → 3 ′ direction.
A 17 bp synthetic DNA was used. 2 μg of mRNA and primer 0.1
μg, and cDNA was synthesized using a cDNA synthesis kit manufactured by Amashiam. Next, using the cDNA cloning system λgt10 also manufactured by Amasiyam,
Was integrated into the EcoRI site of λgt10, and packaged in lambda phage. However, the packaging includes GIGAPACK GOL (Strategene)
D) was used. C60chfl recommended by Strategene
Performing plating using the stock, 10 pieces of square shears,
6,000 phages were formed per sheet, and the phages were transferred to a filter and filtered. As a probe, EcoR I-Xh corresponding to positions 316 to 690 of the nucleotide sequence
o I fragment was used.

56 μg of the aforementioned pCS8, 10 × buffer solution, EcoR I100 unit, Xho I100 unit and water were added to 400 μ,
After stirring, the mixture was incubated at 37 ° C. for 2 hours. After completion of the reaction, electrophoresis was performed on a 1.2% agarose gel to cut out a portion containing the target fragment, and the DNA was electrically extracted as described above to obtain 2.2 μg. 0.1g of this,
Using the multi-prime labeling system described above, the probe was labeled with ³² P to obtain a probe having a specific activity of 2 × 10 ⁸ CPM / μg. Hybridization was performed using 3 × 10 ⁶ CPM of this probe and the previously prepared filter. The filters were then washed and autoradiographed. Since one positive signal was obtained, a secondary screening was performed.
A single plaque was isolated (designated λcs131).

(Preparation of DNA of λcs131) Next, the phage was subjected to the liquid culture method (Gene
Page, Tokyo Kagaku Dojin Publishing Co., Ltd.), and 24 μg of DNA was obtained therefrom.

(Identification of Insert Fragment) 1 μg of the DNA obtained above, 3 μ of 10 × buffer, EcoR I30
Add the unit to make the total volume 30μ, stir and then 37 ℃
For 2 hours. The reaction solution was analyzed on a 1.2% agarose gel, and a 0.5 kb insert was identified.

(Determination of base sequence of inserted fragment) 20 μg of the previously obtained phage DNA and 60 μl of 10 × buffer solution
And EcoR I100 unit to make a total volume of 600 μm, and after stirring, reacted at 37 ° C. for 2 hours. The reaction solution was concentrated by ethanol precipitation, and the DNA was dissolved in 40 µ of TE. This was subjected to 1.2% agarose gel electrophoresis, a portion containing the target insert was cut out, and DNA was electrically extracted to obtain 100 ng. 50 ng of the obtained DNA fragment and RFM13mp1
50 ng of 9 EcoRI digests, 1 μl of 10 × ligation buffer
, 10 [mu] added and T ₄ DNA Torigaze 2.8 Yunitsuto and water
After stirring, the mixture was incubated at 15 ° C. overnight.

Using 1 μl of the ligation reaction solution, the lower part of JM109 was transformed, and a recombinant clone was obtained in the same manner as described above. This clone was used to obtain 0.
The nucleotide sequence of the 5 kb insert was determined. The region on the physical map covered by the inserted fragment is shown in FIG. 1 as λcs131. From the results of the nucleotide sequence analysis in Examples (1-1) and (1-2), a part of the functional unit L of human calpastatin and the entire nucleotide sequence and amino acid sequence of functional units 1 to 4 were determined. Was. The results are shown below.

Example 2 Construction of Expression Plasmid (2-1) Construction of Plasmid Expressing Polypeptide Containing Functional Units 1, 2, 3 and 4 Sequences overlap λcs19 and λcs131 inserted EcoRI fragments by the following procedure. Ac in the area
The expression plasmid (pHCS12) was inserted into the NcoI site of PUC119 (N) in frame.
1).

After digesting pCS860μ with EcoRI, ethanol precipitation, add 10 × buffer solution 45μ, Acc I270 unit and water, stir to a total volume of 450μ, stir at 37 ° C for 2.5 minutes, then add 270μ 0.2M EDTA. In addition, the reaction was terminated, and the plasmid DNA was partially digested with AccI. Next, electrophoresis was performed on a 1% agarose gel to obtain 1.77 kb.
The portion containing the EcoR I-Acc I partial digestion fragment of
NA was extracted electrically (actually, 4 μg was obtained in a mixture with the undigested EcoRI fragment). 2 μg of this partially digested fragment
BamHI-AccI fragment cut out from λcs131 and prepared.
3 μg pUC119 (N) was double-digested with EcoR I and BamHI, 0.2 μg, 2 × 10 × ligation buffer and water were added, and the mixture was stirred at 15 ° C. overnight with a total volume of 20 μg.

After inactivating the ligase by keeping the reaction solution at 70 ° C for 10 minutes
Remove 10μ, add 5μ of 10 × TA buffer, Sma I1
0 units and water were added to make the total amount 50 μ, and the mixture was incubated at 37 ° C. for 1 hour. The lower part of JM109 was transformed using 15 μ of these to obtain pHCS13. pHCS13 plasmid DN
A was prepared, and 0.8 μg of DNA was digested with BamHI, followed by phenol extraction and ethanol precipitation to recover the DNA. Then a 10 × buffer (70 mM Tris HCl, pH 7.5, 200 mM NaCl, 70 mM
mM MgCl ₂ ) 1.5μ, Klenow enzyme 2 units, dNTP
A mixed solution (1 mM each) and water were added, and the mixture was stirred at a total volume of 15 μm and incubated at 37 ° C. for 10 minutes.

Next, a 12-mer Nco I linker [d (pCAGCCATGGCT
H)] 0.1 μg, 200 mM dTT 1 μ, 10 mM ATP 0.5 μ, T ₄ D
2.8 units of NA ligase and water were added, and the mixture was stirred at a total volume of 20 μm and incubated at 15 ° C. overnight.

Sepharose CL-4B column with 1.5 ml bed volume (Pharmacia, 0.15 M NaCl, Tris-HCl (pH 7.5), 0.1 mM
After removing the excess linker by EDTA (equilibrated with EDTA), MgCl ₂ was added to a final concentration of 10 mM, and 100 units of NcoI was further added to make the total amount 550 μm, followed by incubation at 37 ° C. for 2.5 hours. Next, keep at 70 ° C for 10 minutes to inactivate Nco I,
Taken out 250μ from the reaction system, which in addition to ATP and DTT as respectively a final concentration 0.2 mM, in 10 mM, further adding T ⁴ DNA ligase 15 Yunitsuto, overnight Inkiyubeto at 15 ° C..
Next, the reaction solution was kept at 70 ° C. for 10 minutes to inactivate the ligase, and 5 μM NaCl (5 μl) and Sal I50 unit were added.
After incubation for a period of time, the mixture was concentrated with a concentrator and dialyzed against water (final volume: 63 µ). From this, 25 μl was taken out and the lower part of JM109 was transformed to obtain pHCS121. FIG. 2 shows the construction process as a process diagram.

Escherichia coli JM109 with pHCS121
M109 / pHCS121 and deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology [Microtechnical Laboratory No. 9989 (FERM P-998)
9)].

(2-2) Construction of Plasmid Expressing Functional Unit 3 Maki et al.'S method [Feves Letters, Vol. 223, No. 174-
180 (1987)], an NcoI site was introduced into the portion of functional unit 2 in contact with functional unit 3 (1079 to 1084 by the nucleotide number) using pCS8. Ie pCS8
The Nco I site containing the start codon of functional unit 3 is introduced by a site-specific in vitro mutant preparation system (Amasiyam) and a partial specific mutagenesis operation using synthetic DNA primers. The resulting plasmid was obtained. N
After digestion of the plasmid into which the coI site had been introduced with NcoI + XbaI, the fragment was cut out by 1% agarose gel electrophoresis, and the fragment was electroeluted and recovered. A plasmid was prepared by inserting this fragment into the NcoI-XbaI site of pUC119 (N) in the same manner as described above. Next, a site-directed mutagenesis operation was performed in the same manner as described above, and an Afr II site was added to the C-terminal portion of the functional site 3 (from nucleotides 1475 to 1480).
That is, a stop codon was introduced. FIG. 3 shows the construction process as a process chart. This plasmid was named pHCS111, and E. coli JM109 carrying pHCS111 was replaced with Escherichia coli.
It was designated as JM109 / pHCS111 and deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology [Microtechnical Laboratory No. 9988 (FERM P-998).
8)].

Example 3 Expression of human calpastatin-like polypeptide in Escherichia coli (3-1) Expression of functional unit 3 Escherichia coli JM109 / pHCS111 obtained in Example 2
(FERM P-9988) was inoculated in 5 ml of L medium and cultured with shaking at 37 ° C. When the medium reached mid-logarithmic growth, IPTG was added to a final concentration of 2 mM, and the cells were further cultured for 4 hours. After culturing, the cells were cooled to 0 ° C., NaN ₃ was added to a final concentration of 0.02%, stirred, and then collected. The supernatant was discarded, and the cells were washed with 0.6 ml of M buffer [20 mM Tris HCl (pH 7.5), 1 mM
Washed with EDTA, 1 mM EGTA, 0.2 mM PMSF, 5 mM 2-mercaptoethanol], centrifuged, suspended in 0.5 ml of M buffer, and added lysozyme to a final concentration of 0.5 mg / ml.
Incubate at 0 ° C for 10 minutes. Next, freeze-thaw (−70
20 ° C) 3 times, and centrifuged at 14,000 rpm for 20 minutes.
The supernatant was kept at 95 ° C for 10 minutes. Further increase this to 14,000 rpm for 10
After centrifugation for minutes, the supernatant was collected. The calpastatin activity of the preparation thus obtained was measured by observing the inhibition of calpain as shown below (Journal of Biochemistry, vol. 95, p. 1759 (1984)).
The sample, calpain, 40% of 2% casein, 20 μ of a 6 mg / ml cysteine solution and water were mixed to make a total volume of 180 μ. Next, after preheating at 30 ° C. for 5 minutes, 20 μm of 50 mM calcium chloride was added and mixed, and the mixture was incubated at 30 ° C. for 30 minutes. Next, 200 µ of 5% TCA was added to stop the reaction.
After centrifugation at pm for 10 minutes, the supernatant was collected.

Take 0.2 ml of this solution and add a monoiodoacetic acid solution (0.7 M Na
100 μl of a mixture of CO ₃ /0.1N NaOH and 2N monoiodoacetic acid / 2N NaOH in a ratio of 1: 1 was added, and a Lowry reagent (0.5% Cu
SO ₄ and 0.1% tartaric acid potassium, a _{2% Na 2 CO 3 /0.1N NaOH} 1: 1: 50
1 ml) was added, mixed, and allowed to stand for 10 minutes.

Then stirred immediately by addition of 1N phenol reagent 100 microns, to measure the A ₇₅₀ nm after at least 30 minutes, it was calculated than this calpain inhibitory activity.

10 μm of the preparation obtained by the above method inhibited pig erythrocyte calpain I by 66% in a 0.2 ml calpain reaction system. Next, the polypeptide that was the active form was purified, and it was confirmed that the structure was a polypeptide containing the structure represented by D3. In addition, Escherichia coli JM109 which does not carry the plasmid
Was treated in the same manner, but no inhibitory activity was observed.

(3-2) Expression of polypeptide containing functional units 1, 2, 3, and 4 Escherichia coli JM109 / pHCS1 constructed in (2-1)
Using 21 (FERM P-9988), the same operation as that for expressing and preparing the protein of the above functional unit 3 was performed, and the calpain inhibitory activity was confirmed in the prepared solution. Next, the polypeptide that is the active form is purified, and its structure is D1, D2, D3, and D4.
It was confirmed that the polypeptide contained.

〔The invention's effect〕

From the above results, the present invention has revealed the amino acid sequence necessary for expressing the activity of human calpastatin and its DNA sequence, and provided a method for producing a human calpastatin-like polypeptide by genetic engineering.

[Brief description of the drawings]

FIG. 1 is a diagram showing a restriction enzyme map of human calpastatin cDNA and a functional unit structure, and FIG. 2 is a diagram showing a polypeptide having a part of functional unit L of human calpastatin and an amino acid sequence of functional units 1 to 4. FIG. 3 is a flow chart showing a construction process of a recombinant plasmid for expression, and FIG. 3 shows a construction process of a recombinant plasmid for expressing a polypeptide having the amino acid sequence of functional unit 3 of human calpastatin. It is a process drawing.

Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication // A61K 38/55 ABJ A61K 37/64 ABJ ABL ADU ABU ABU ABL ADU ABU (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Shoichi Hatanaka 1-32 Takano Higashikaimachi, Sakyo-ku, Kyoto, Kyoto Prefecture 35-203, Higashioji Takano 3rd House 35-203 (72) Inventor, Masatoshi Maki Gokasho, Uji-shi, Kyoto Kyoto University Staff Dormitory 655 (56) Reference Proc. Natl. Acad. Sc i. VSA 84 (1987) p. 3590-3594 FEBS Ieiters 223 [1] (1987) p. 174−180

Claims (4)

(57) [Claims] 1. A polypeptide represented by the following formula D1, D2, D3 or D4, which is a functional unit of human calpastatin, or a human calpastatin-like polypeptide to which the following formulas D1, D2, D3 and D4 are bound in this order: . 2. A nucleic acid encoding the polypeptide according to claim 1. 3. A transformant into which a recombinant plasmid containing the nucleic acid according to claim 2 has been introduced. 4. A method for producing a human calpastatin-like polypeptide, which comprises culturing the transformant according to claim 3, and collecting the human calpastatin-like polypeptide according to claim 1 from the culture.