JP3012931B1 - SPARC fusion protein and method for producing the same - Google Patents

Abstract

The present invention relates to a SPARC fusion protein obtained by fusing SPARC and thioredoxin, a DNA encoding the protein, and a transformant obtained by introducing a recombinant vector containing the DNA into a host cell. And producing a protein encoded by the DNA in the culture, accumulating the protein in the culture, and collecting the protein from the culture. According to the present invention, a SPARC protein, which has various physiological activities such as angiogenesis, anti-cell adhesion activity, ECM reconstitution, and bone formation, is expected to be used as a research reagent or pharmaceutical because of its various physiological activities. As a soluble fusion protein that does not lose its physiological activity and is easily prepared as a medicament, it can be efficiently and inexpensively prepared in large quantities.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a SPARC fusion protein and a method for producing the same.

[0002]

[Prior art] SPARC (secreted protein acidic and ric
h in cysteine; “acidic and cysteine-rich secreted protein”) was first reported as osteonectin, the major non-collagenous protein present in bone (Termine, JD et al., Cell, 26). :
99-105, 1981), which has attracted attention as a functional bone-specific protein because it has strong binding properties to collagen and hydroxyapatite and has Ca ²⁺ binding ability. This SPARC is
Molecular weight 43 extracellularly secreted with the growth of adherent cultured cells
The gene sequence was determined from that reported as the kD protein (Sage.H. et al., J. Biol. Chem., 259, 3993-4
007, 1984) and BM-40 (Mann, K. et al., FEBS Lett., 218:
162-172, 1987). SPARC was subsequently secreted from various cultured cells, and in particular, remarkable secretion from normal fibroblasts and vascular endothelial cells (Lan
eTF & Sage, EH, FASEB J., 8: 163-173, 1994).

[0003] As for the function of SPARC, it is known that addition of SPARC to a cell culture system inhibits cell adhesion to extracellular matrix molecules in a concentration-dependent manner. adhesive pro
tein "(Sage, EH & Bornstein, P., J. Bio
l. Chem., 266: 14831-14834, 1991). This action does not competitively inhibit the binding of integrin to cell adhesion ligands, but by binding to SPARC cell surface receptors,
It is reported to act on the cytoskeletal system such as vinculin via intracellular signal transduction to degrade the focal adhesion plaque (Murphy-Ullrich, JE et al.,
J. Cell. Biochem., 57: 341-350, 1995). Also, SPARC
The most notable features of ECM (extracellular m
atrix; extracellular matrix) production, and increase ECM-degrading protease production (Lane.TF &
Sage, EH, FASEB J., 8: 163-173, 1994), which is highly expressed in angiogenesis sites and reduces the secretion of fibronectin and thrombospondin in endothelial cells.
Therefore, it is thought that SPARC performs ECM reconstitution by linking these actions, and in particular, promotes cell growth control and increased intercellular permeability in vascular endothelium, and is essential for cell growth and angiogenic activity. ing. SPARC having various physiological activities as described above is noted for its usefulness as a therapeutic agent, and clinical application is expected.

Until now, attempts have been made to produce SPARC by genetic engineering techniques using animal cells as host cells. However, the yield is very low and the yield cannot be satisfactory. There was a problem that several steps were required for purification, and very labor and cost were required. In some cases, Escherichia coli was used as the host cell, but SPARC did not appear in the soluble fraction, or it appeared in the soluble fraction in an extremely small amount, and SPARC accumulated in the inclusion body that was the insoluble fraction. Is an inactive form that does not retain physiological activity, and no method has been established to obtain a soluble, active form of SPARC by folding the SS bond of inactive SPARC isolated and purified from the insoluble fraction into an appropriate form In addition, even when expressed as a fusion protein such as glutathione-S-transferase (GST), the protein accumulates in inclusion bodies.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to an SP which has various physiological activities and is therefore highly useful as a therapeutic agent.
The aim is to produce ARC efficiently and inexpensively and in large quantities.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, incorporated a SPARC encoding gene downstream of a thioredoxin encoding gene, and expressed the gene in Escherichia coli. As a result, the present inventors have found that the protein can be produced in a large amount as a soluble protein which is easy to formulate without losing the physiological activity of SPARC, and have completed the present invention.

That is, the first invention of the present invention is as follows:
The protein shown in (a) or (b). (a) a protein having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing; And a protein having the physiological activity of SPARC. The deletion, substitution or addition of the above amino acids can be carried out by a site-directed mutagenesis method which is a well-known technique before filing, and one or several amino acids are deleted by the site-directed mutagenesis method. It means the number of amino acids that can be lost, substituted or added.

[0008] The physiological activity of the SPARC is
Angiogenesis, anti-cell adhesion activity (cell spreading inhibitory activity and cell sphering activity), extracellular matrix reconstitution (decreased extracellular matrix production and increased extracellular matrix degrading protease production), and bone formation (mineralization) ). The second invention of the present invention relates to a DNA encoding the protein or a DN consisting of the nucleotide sequence from the 5209th to the 6609th nucleotide of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing.
About A.

[0009] A third invention of the present invention is to transform a transformant obtained by introducing a recombinant vector containing the above-mentioned DNA into a host cell in a medium, and to prepare a protein encoded by the DNA in a culture. A method for producing a SPARC fusion protein, comprising producing and accumulating the protein, and collecting the protein from the culture. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The SPARC fusion protein in the present invention is a fusion protein of SPARC and thioredoxin. This SPARC fusion protein uses, for example, a commercially available expression vector containing a thioredoxin gene,
It can be prepared by inserting a SPARC-encoding DNA (hereinafter referred to as a SPARC gene) into a multicloning site downstream of the thioredoxin gene and expressing it in E. coli or the like in a large amount. Hereinafter, this preparation is specifically described.

[1] Cloning of SPARC Gene (1) Amplification of SPARC cDNA Fragment In the present invention, the cloning of SPARC gene is performed by RT-PC
Perform by the R method. First, total RNA is prepared from a tissue or cell expressing SPARC, for example, mouse cerebral cortex. All R
As a method for preparing NA, guanidine acid thiocyanate-phenol-chloroform (AGPC) method (P. Chomczyn)
ski and N. Sacchi, Analytical Biochemistry, 162: 1
56, 1987), guanidine thiocyanate-cesium trifluoroacetate method (H. Okayama et al., Methods in Enzymol
ogy, 154: 3, 1987). Using this total RNA, the method of J. Sambrook et al. (J. Sambrook et al., Molecula
rCloning, A Laboratory Manual, Second Edition, Col
d Spring Harbor Laboratory Press, 1989) and the like, and the mRNA in the RNA is converted to cDNA by performing a reverse transcriptase reaction. Next, using this cDNA as a template, the base sequence of the previously reported SPARC cDNA (JH McVey et al., Journey
nal of Biochemistry, 263: 11111-11116, 1988), and PCR was performed using SPARC cDNA.
Amplify the fragment.

(2) Determination of Base Sequence of Amplified Fragment The base sequence of the amplified fragment is determined by a cycle sequence method. First, the fragment amplified in (1) is
escript T-vector, pBluescriptII T-vector, pT7B
lue T-vector, pT7Blue-2 Subcloned into a vector for TA cloning such as T-vector, T7 primer, T3 primer, M13 (-20) primer, reverse primer, SK primer, KS primer, R-20mer primer, U In the presence of a primer such as a -19-mer primer, the template DNA is heat-denatured, the primer is annealed, and the sequencing reaction with fluorescently labeled ddNTP is made one cycle, and this cycle is repeated about 30 cycles. The sequencing reaction can be performed using a thermostable polymerase such as Taq polymerase and a commercially available thermal cycler for PCR.

[2] Preparation of Plasmid Vector for Expression of SPARC Fusion Protein In the present invention, plasmid vectors that can be used for expression of a SPARC fusion protein include those capable of replicating in a host cell, an appropriate transformation marker gene,
For example, having the ampicillin resistance gene, kanamycin resistance gene, tetranecycline resistance gene, etc., under the control of a promoter that functions in the introduced host cell, in the state fused with thioredoxin when the SPARC gene is connected downstream of the thioredoxin gene Any substance that satisfies the condition that it can express SPARC, and desirably an induction vector that has a low expression level in the absence of an inducer and a high expression induction level. May be. In addition, it is desirable to have an appropriate restriction enzyme site for inserting thioredoxin-SPARC cDNA. As such a vector, when the host cell is Escherichia coli, for example, pET vector (Novagen), pTrxFUS vector (Invitrogen)
, PCYB vector (manufactured by NEW ENGLAMD Bio Labs), etc., when the host cell is yeast, for example, pESP-1 expression vector
(STRATAGENE), pAUR123 vector (Takara Shuzo),
pPIC vector (manufactured by Invitrogen) or the like, or when the host cell is an animal cell, for example, a pMAM-neo expression vector
(Manufactured by CLONTECH), pCDNA3.1 vector (manufactured by Invitrogen)
, PBK-CMV vector (manufactured by STRATAGENE) or the like, when the host cell is an insect cell, for example, pBacPAK vector (CLO
NTECH), pAcUW31 vector (CLONTECH), pAcP
(+) IE1 vector (Novagen) and the like.

[0014] The promoter is not particularly limited as long as it can function in each of the host cell for introducing the vector, when Escherichia coli is used as a host cell, for example, T7 promoter, T5 promoter, .lambda.P _L promoter, trp When yeast is used as a host cell, for example, PGK promoter, ADH1 promoter, Gal1-Gal10
Promoter, PHO5 promoter and the like, when using animal cells as host cells, for example, SV40 promoter,
When CMV promoter, CAG promoter, SRα promoter, EF1α promoter, AML promoter, MMTV promoter, MTII promoter, etc. are used as insect cells as host cells, for example, P10 promoter, polyhedron promoter, etc. are exemplified.

A commercially available expression vector into which a thioredoxin gene has been inserted under the control of an appropriate promoter may be used. For example, a pET32a (+) vector (manufactured by Novagen) having a T7 promoter is preferable. Used for SPARC cDNA is added to the above expression plasmid vector.
The fragment was inserted into the restriction enzyme site at the C-terminus of the thioredoxin gene so that the protein frame matched it.
NA can be obtained.

A transformant is prepared by transforming a host cell with the obtained recombinant DNA. In the present invention, Escherichia coli, Bacillus subtilis, yeast, animals, insect cells and the like can be used as host cells for producing a SPARC fusion protein, but Escherichia coli is preferred. Specifically, Escherichia coli JM109, HB101, BL21, Nova
Blue, AD494, B834, HMS174, BLR and the like can be used. It is also preferable to use a protease-deficient strain such as a lon gene-deficient strain among the above strains. Transformation is performed by, for example, the calcium chloride method, electroporation method, rubidium chloride method, lipofection method, DEAE-dextran method, lithium method, spheroplast method, virus and the like.

[3] Production of SPARC fusion protein The transformant obtained as described above is cultured in a medium,
The SPARC fusion protein of the present invention can be obtained by producing and accumulating the protein in the culture and collecting the protein. The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing the host cell.

A culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant. The carbon source may be any as long as the organism can assimilate,
Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, organic acids such as propionic acid, ethanol,
Alcohols such as propanol can be used.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, and corn steep. Liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used. As inorganic substances, potassium monophosphate, potassium diphosphate,
Magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

Specifically, LB medium, H medium, SOB medium,
SOC medium, NZYM medium, TB medium, YT medium, tryptone medium, TYGPN medium, λ medium, super broth medium, M9 medium, M63 medium, A medium and the like can be used. Medium p
It is appropriate that H is adjusted to 6 to 8. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. Culture is 4-40
° C, preferably at 36 to 38 ° C, for 1 to 24 hours, preferably 2 to 24 hours.
The reaction may be performed for 3 hours, and if necessary, ventilation or stirring may be added.

[0021] If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-
When culturing a microorganism transformed with β-D-thiogalactopyranoside or the like using an expression vector using a trp promoter, indoleacrylic acid or the like may be added to the medium.

As a medium for culturing a transformant obtained by using an animal cell as a host, a commonly used medium is used.
RPMI 1640 medium, Eagle's MEM medium, DMEM medium, or the like, or a medium obtained by adding fetal bovine serum or the like to these mediums can be used. In order to isolate and purify the expressed protein from the culture of the above-mentioned transformant, a usual protein isolation and purification method may be used.

Since the SPARC fusion protein of the present invention is expressed in a dissolved state in the cytoplasm, the cells are recovered by centrifugation after suspension of the culture, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, and the like. The cells are crushed with a Manton-Gaurin homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Diethylaminoethyl (DEAE)-Sepharose, anion exchange chromatography using isoresin, S-
Cation exchange chromatography using a resin such as Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose, phenyl sepharose, gel filtration using a molecular sieve, His
A purified sample can be obtained by using an affinity chromatography method using Bind resin (manufactured by Novagen), a chromatofocusing method, or an electrophoresis method such as isoelectric focusing alone or in combination. Also,
The resulting SPARC fusion protein was identified as enterokinase (En
Only SPARC can be cut out with terokinase) or thrombin (Thrombin).

[0024]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. [Example 1] (Preparation of plasmid vector for expression of SPARC fusion protein) (1) Preparation of T vector The method of D. Marchuk et al. (Nucleic Acids Research, 19: 1)
154, 1990). Plasmid Bluescript SK (-) (1
0 μg / μl) (STARATAGENE) 1.0 μl, 10 × buffer H
(Takara Shuzo Co., Ltd.) 2.0 μl, EcoRV (10 U / μl) 2.0 μl, and sterile water 15.0 μl were mixed to adjust the total volume to 20.0 μl.
The reaction was allowed to proceed for a period of time to completely cut the plasmid. To the precipitate formed by ethanol precipitation, 4.0 μl of 10 × PCR buffer (manufactured by PE Applied Systems), 8.0 μl of 10 mM dTTP, Ampli
2.0 μl of Taq DNA polymerase (5 U / μl) (manufactured by PE Applied Systems) and 24.0 μl of sterilized water were added to adjust the total volume to 40.0 μl, and reacted at 70 ° C. for 2 hours. After phenol chloroform extraction and purification and concentration by ethanol precipitation, the resulting precipitate was dissolved in 20 μl of sterilized water to obtain a T vector solution. The T vector was stored at 4 ° C.

(2) Extraction of total RNA from mouse cerebral cortex The method of P. Chomczynski et al. (Analytical Biochemistry,
162: 156-159, 1987). After the mouse cerebral cortex was removed, it was frozen with liquid nitrogen and crushed with a hammer to form a powder. 75 mg of this powder in guanidine solution
(4 M guanidine thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarcosyl sodium, 0.1 M mercaptoethanol) dissolved in 500 μl, and 50 μl of 2 M sodium acetate solution (pH 4.0), DEPC water-saturated phenol (pH 4.0 )
After adding 50 μl and 100 μl of chloroform and stirring,
Left at ℃ for 15 minutes. After centrifugation at 10,000 g for 20 minutes, the aqueous layer was recovered, and 600 μl of the same volume of cooled isopropanol and 2 μl of glycogen (20 μg / ml) were added thereto.
It was left at 0 ° C. for 1 hour. The precipitate formed by centrifugation at 16,000 g for 10 minutes was completely dissolved in 500 μl of a guanidine solution, and the above process was repeated again. To the precipitate generated by isopropanol precipitation, 50 μl of DEPC water was added and dissolved to obtain a total RNA solution. Finally from mouse cerebral cortex 0.3g to 2
54 μg of total RNA was obtained.

(3) Cloning of mouse SPARC gene by RT-PCR (3-1) Synthesis of single-stranded cDNA (2)
RNA (2.5 μg / μl) 1.2 μl with random primer (100 ng)
/ μl) 1.0 μl, 2.8 μl of DEPC-treated sterilized water to add a total volume of
It was adjusted to 0 μl and subjected to a heat denaturation treatment at 70 ° C. for 10 minutes. 2.0 μl of 5 × buffer (GIBCO BRL), 2.0 μl of 0.1 M dithiothreitol, 8.0 μl of 1.25 mM dNTP, SUPERSCRI
The total volume was adjusted to 20.0 μl by adding 1.0 μl of PT ^™ II Reverse Transcriptase (200 U / μl), and reacted at 42 ° C. for 2 hours. The reverse transcriptase was completely inactivated by heating at 70 ° C. for 15 minutes to obtain a single-stranded cDNA solution.

(3-2) PCR reaction 10 × PCR buffer was added to 5.0 μl of the single-stranded cDNA solution obtained in (3-1).
(Manufactured by PE Applied Systems) 2.5 μl, Ampli Taq DNA
polymerase (5U / μl) (manufactured by PE Applied Systems) 0.25 μl, 1.25 mM dNTP 8.0 μl, 60 ng / μl Primer 1 (5′-CCGAGAGTTCCCAGCATCAT-3 ′; 20mer / SEQ ID NO: 3 in Sequence Listing) and Primer 2 (5 '-TCAAACCAATTCACC
AGTCT-3 '; 20mer / SEQ ID NO: 4)
l, 4.25 μl of sterilized water was added to adjust the total volume to 25.0 μl, and the mixture was used as a reaction solution. Next, DNA Thermal cycler PJ2000 Model
PCR using 480 (PE Applied Systems) (denaturation reaction; 94 ° C., 30 seconds, annealing reaction; 50 ° C., 45 seconds, synthesis reaction; 72 ° C., 90 seconds; 35 cycles), followed by 10 minutes at 72 ° C.
The extension reaction was performed for minutes. A PCR product of about 1750 bp was separated and purified by 1% agarose gel electrophoresis, and recovered from the gel using an ultra-free MC filter (Millipore). The recovered PCR product is DNA Ligation Kit Ver.
2 (manufactured by Takara Shuzo) and subcloned into the T vector. The primer was a mouse SPARC cDNA base sequence (JH McVey et. Al. Journal of Biochemistry 26).
3, 11111-11116, 1988). Primers 1 and 2 are oligonucleotide sequences corresponding to the SPARC cDNA nucleotide sequences 72-91 and 1801-1820, respectively.

(3-3) Determination of cDNA base sequence of PCR product T vector (pB
SK-SPARC vector)
The NA base sequence was determined. Using 1.5 μg of the above T vector purified by QIAGEN plasmid Minikit (Funakoshi) as a template, PRISM Ready Reaction Terminator Cy
Sequence samples were prepared using the cle Sequencing Kit (manufactured by PE Applied Systems). PCR is DNA
Using Thermalcycler PJ2000 Model 480 (PE Applied Systems), denaturation reaction: 96 ° C, 30 seconds, annealing reaction: 50 ° C, 15 seconds, synthesis reaction; 60 ° C, 240 seconds; 25 cycles were performed. After removing unreacted products by ethanol precipitation, the PCR product is dissolved in 4.0 μl of a denaturing solution (5 mg / ml blue dextran, 8.3 mM EDTA pH 8.0, 83.3% formamide) and subjected to heat denaturation treatment at 90 ° C. for 2 minutes. Was. Immediately thereafter, the cDNA base sequence was analyzed using the ABI 373S sequencer (manufactured by PE Applied Systems), and the PCR product was used as the mouse SPARC cDNA base sequence (72-1820).
Was confirmed.

(4) Subcloning into pGEX 4T-1 vector pBSK-SPARC vector (6.25 μg / μl) prepared in (3)
Add 10 × buffer 4 (NEB) 10 μl, BamHI 2 μl, Xho
Add 2 μl of I (12 U / μl) and 76 μl of sterile water to make a total volume of 100
μl and reacted at 37 ° C. for 2 hours. BamHI-X of about 1800bp
The hoI inserted fragment was separated and purified by 1% agarose gel electrophoresis, recovered from the gel using GENE CLEAN II kit (manufactured by Funakoshi), and TE solution (Tris-HCl pH 8.0, 0.5 mM E
DTA pH 8.0). Next, this BamHI-Xh
oI Insert Fragment Solution 10 × Buffer 4 (NEB) 10 μl in 42 μl
l, BspHI (10 U / μl) 2 μl, SmaI (10 U / μl) 2 μl,
The total volume was adjusted to 100 μl by adding 44 μl of sterilized water, and the reaction was carried out at 37 ° C. for 2 hours. The BspHI-SmaI insert of about 1500 bp was separated and purified by 1% agarose gel electrophoresis, followed by GENE CLEAN I
Collect from the gel using I kit (Funakoshi) and extract TE solution
Dissolved in 48 μl. This BspHI-SmaI insert (960 ng)
Was blunt-ended using DNA Blunting Kit (Takara Shuzo) and dissolved in 10 μl of TE solution to obtain a blunt-ended insert fragment solution.

On the other hand, the pGEX-4T1 vector (500 ng / μl) (Ph
armacia Biotech) 10 μl buffer 4 (NEB) in 2 μl
2 μl, 1 μl of SmaI (10 U / μl) and 15 μl of sterilized water were added to make a total volume of 20 μl, and reacted at 37 ° C. for 2 hours. After phenol chloroform extraction and purification and concentration by ethanol precipitation, 5 μl of 10 × dephosphorylation buffer (Takara Shuzo Co., Ltd.) and calf small intestine-derived alkaline phosphatase (10
(U / μl) 1 μl and 44 μl of sterile water were added to make a total volume of 50 μl, and the mixture was reacted at 37 ° C. for 15 minutes for dephosphorylation.
After a heat treatment at 52 ° C. for 15 minutes to deactivate the phosphatase, purification and concentration by enol chloroform extraction and ethanol precipitation were performed. The resulting precipitate is a TE solution 20
The resultant was dissolved in μl to obtain a dephosphorylated pGEX-4T1 vector solution.

Finally, after mixing 20 μl of the dephosphorylated pGEX-4T1 vector solution and 10 μl of the blunt-ended insert fragment solution, the mixture was concentrated by ethanol precipitation, and the DNA Ligation Kit V
er.2 (manufactured by Takara Shuzo Co., Ltd.) to ligate the BspHI-SmaI blunt-ended insert to the Smal site of the pGEX-4T1 vector
SPARC vector was obtained. In addition, the fact that the BspHI-SmaI blunt-ended insert fragment was incorporated into the pGEX-4T1 vector as planned, means that the BamHI-XhoI insert fragment of the pGEX-4T-SPARC vector was converted into a pBluescript SK (-) vector (manufactured by STRATAGENE). Bam
It was confirmed by subcloning into the HI-XhoI site and performing cDNA base sequence analysis.

(5) Preparation of pET32a (+)-SPARC vector pGEX-4T-SPARC vector (0.25 μg / μl) prepared in (4)
10 μl of 10 × buffer K (Takara Shuzo) in 40 μl, BamHI (1
0 U / μl) 2 μl, XhoI (12 U / μl) 2 μl, sterile water 46 μl
Was added to make the total volume 100 μl, and the mixture was reacted at 37 ° C. for 2 hours.
The BamHI-XhoI inserted fragment was separated and purified by 1% agarose gel electrophoresis, and the BamHI-XhoI inserted fragment was recovered from the gel using a GENE CLEAN II kit (Funakoshi), and the TE solution 4
Dissolved in 8 μl. It was subcloned into the BamHI-XhoI site of the pET32a (+) vector (Novagen). The finally obtained pET32a (+)-SPARC vector was analyzed for cDNA base sequence to confirm that the gene reading frame was in agreement.
The entire cDNA base sequence of the pET32a (+)-SPARC vector is shown in SEQ ID NO: 2, and the entire amino acid sequence of the SPARC fusion protein produced by this vector is shown in SEQ ID NO: 1 and FIG. FIG. 2 shows the outline of the construction of the pET32a (+)-SPARC vector.

[Example 2] (Production of SPARC fusion protein) Escherichia coli AD494 (DE3) was transformed with the pET32a (+)-SPARC vector prepared in Example 1. The Escherichia coli was cultured in an LB solution containing 30 μg / ml kanamycin at 37 ° C. for 3 hours until OD ₅₆₀ = 0.6, and then incubated at 20 ° C. in the presence of 1 mM IPTG for 6 hours.
Shaking culture was performed for hours. Cells are centrifuged (6,000 × g, 3
0 minutes) and buffer A (5 mM Imidazole, 500 mM
The suspension was suspended in 20 ml of NaCl, 20 mM Tris-HCl, pH 7.9), sonicated, and the soluble fraction was collected by centrifugation (30,000 × g, 30 minutes). After adding this soluble fraction to a His Bind Resin column (Novagnen), buffer B (1M Imidazol) was added.
e, The adsorbed fraction was eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.9). The eluate was concentrated to about 1 ml, and buffer C (10 mM
Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% NP-40) dialyzed overnight, and gel filtration column [Superdex 75 (Pharmacia)]
Was added. Buffer D (10 mM Tris-HCl pH 7.4, 100 mM N
aCl) at a flow rate of 1 ml / min to fractionate every 5 ml,
A single peak (fractions 8-10) with a retention time of 40 minutes was collected and dialyzed against buffer B overnight. After separating this sample with 10% SDS-pAGE, CBB staining was performed.
It was confirmed that it had the above purity (FIG. 3). The yield was about 1 mg / L of the LB culture solution.

[0034]

[Sequence List] SEQUENCE LISTING <110> Director-General of Agency of Industrial Science and Technology <120> SPARC fusion protein and a process for producing the same <130> 11900252 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 467 <212> PRT <213> Mus musculus <400> 1 Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Gly His Met His His His His His Ser Ser Gly Leu Val Pro 115 120 125 Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln 130 135 140 His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met 145 150 155 160 Ala Asp Ile Gly Ser Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu 165 170 175 Ala Gly Arg Ala Leu Ala Ala Pro Gln Gln Thr Glu Val Ala Glu Glu 180 185 190 Ile Val Glu Glu Glu Thr Val Val Glu Glu Thr Gly Val Pro Val Gly 195 200 205 Ala Asn Pro Val Gln Val Glu Met Gly Glu Phe Glu Asp Gly Ala Glu 210 215 220 Glu Thr Val Glu Glu Val Val Ala Asp Asn Pro Cys Gln Asn His His 225 230 235 240 Cys Lys His Gly Lys Val Cys Glu Leu Asp Glu Ser Asn Thr Pro Met 245 250 255 Cys Val Cys Gln Asp Pro Thr Ser Cys Pro Ala Pro Ile Gly Glu Phe 260 265 270 Glu Lys Val Cys Ser Asn Asp Asn Lys Thr Phe Asp Ser Ser Cys His 275 280 285 Phe Phe Ala Thr Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys 290 295 300 Leu His Leu Asp Tyr Ile Gly Pro Cys Lys Tyr Ile Ala Pro Cys Leu 305 310 315 320 Asp Ser Glu Leu Thr Glu Phe Pro Leu Arg Met Arg Asp Trp Leu Lys 325 330 335 Asn Val Leu Val Thr Leu Tyr Glu Arg Asp Glu Gly Asn Asn Leu Leu 340 345 350 Thr Glu Lys Gln Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys 355 360 365 Arg Leu Glu Ala Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp Phe 370 375 380 Glu Lys Asn Tyr Asn Met Tyr Ile Phe Pro Val His Trp Gln Phe Gly 385 390 395 400 400 Gln Leu Asp Gln His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu 405 410 415 Ala Pro Leu Arg Ala Pro Leu Ile Pro Met Glu His Cys Thr Thr Arg 420 425 430 Phe Phe Glu Thr Cys Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Glu 435 440 445 Glu Trp Ala Gly Cys Phe Gly Ile Lys Glu Gln Asp Ile Asn Lys Asp 450 455 460 Leu Val Ile 465 <210> 2 <211> 7375 <212> DNA <213> Mus musculus <220> <221> CDS <222> (5209) .. (6609) <400> 2 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctatt c 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaa aggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720 gcggtttgcg tattgggcgc cagg gtggtt tttcttttca ccagtgagac gggcaacagc 3780 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560 gccagttgtt gtgccacgcg gttgggaat g taattcagct ccgccatcgc cgcttccact 4620 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacat atg agc gat 5217 Met Ser Asp 1 aaa att att cac ctg act gac gac agt ttt gac acg gat gta ctc aaa 5265 Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys 5 10 15 gcg gac ggg gcg atc ctc gtc gat ttc tgg gca gag tgg tgc ggt ccg 5313 Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro 20 25 30 35 tgc aaa atg atc gcc ccg att ctg gat gaa atc gct gac gaa tat cag 5361 Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln 40 45 50 ggc aaa ctg acc gtt gca aaa ctg aac atc gat caa aac cct ggc act 5409 Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr 55 60 65 gcg ccg aaa tat ggc atc cgt ggt atc ccg act ctg ctg ctg aaa 5457 Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys 70 75 80 aac ggt gaa gtg gcg gca acc aaa gtg ggt gca ctg tct aaa ggt cag 5505 Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln 85 90 95 ttg aaa gag ttc ctc gac gct aac ctg gcc ggt tct ggt tct ggc cat 5553 Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser Gly His 100 105 110 115 atg cac cat cat cat cat cat cat tct tct ggt ctg gtg cca cgc ggt tct 5601 Met His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser 120 125 130 ggt atg aaa gaa acc gct gct gct aaa ttc gaa cgc cag cac atg gac 5649 Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp 135 140 145 agc cca gat ctg ggt acc gac gac gac gac aag gcc atg gct gat atc 5697 Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile 150 155 160 gga tcc atg agg gcc tgg atc ttc ttt ctc ctt tgc ctg gcc ggg agg 5745 Gly Ser Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg 165 170 175 gcc ctg gca gcc cct cag cag act gaa gtt gct gtg gag 5793 Ala Leu Ala Ala Pro Gln Gln Thr Glu Val Ala Glu Glu Ile Val Glu 180 185 190 195 gag gaa acc gtg gtg gag gag aca ggg gta cct gtg ggt gcc aac cca 5841 Glu Glu Thr Val Val Glu Glu Thr Gly Val Pro Val Gly Ala Asn Pro 200 205 210 gtc cag gtg gaa atg gga gaa ttt gag gac ggt gca gag gaa acg gtc 5889 Val Gln Val Glu Met Gly Glu Phe Glu Asp Gly Ala Glu Glu Thr Val 215 220 225 gag gag gtg gtg gct gac aac ccc tgc cag aac cat cat tgc aaa cat 5937 Glu Glu Val Val Ala Asp Asn Pro Cys Gln Asn His His Cys Lys His 230 235 240 ggc aag gtg tgt gag ctg gac gag agc aac acc ccc atg tgt gtg tgc 5985 Gly Lys Val Cys Glu Leu Asp Glu Ser Asn Thr Pro Met Cys Val Cys 245 250 255 cag gac ccc acc agc tgc cct gct ccc att ggc gag ttt gag aag gta 6033 Gln Asp Pro Thrr Ser Cys Pro Ala Pro Ile Gly Glu Phe Glu Lys Val 260 265 270 275 tgc agc aat gac aac aag acc ttc gac tct tcc tgc cac ttc ttt gcc 6081 Cys Ser Asn Asp Asn Lys Thr Phe Asp Ser Ser Cys His Phe Phe Ala 280 285 290 acc aag tgc acc ctg gag ggc acc aag aag ggc cac aag ctc cac ctg 6129 Thr Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys Leu His Leu 295 300 305 gac tac atc gga cca tgc aaa tac atc gcc ccc tgc ctg gat tcc gag 6177 Asp Tyr Ile Gly Cys Lys Tyr Ile Ala Pro Cys Leu Asp Ser Glu 310 315 320 ctg acc gaa ttc cct ctg cgc atg cgt gac tgg ctc aaa aat gtc ctg 6225 Leu Thr Glu Plu Pro Leu Arg Met Arg Asp Trp Lep Lyu Asn Val Leu 325 330 gtc acc ttg tac gag aga gat gag ggc aac aac ctc ctc act gag aag 6273 Val Thr Leu Tyr Glu Arg Asp Glu Gly Asn Asn Leu Leu Thr Glu Lys 340 345 350 355 cag aag ctg cgt gtg aag aag atc cat gag aac ag aag cgc ctg gag 6321 Gln Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys Arg Leu Glu 360 365 370 gct gga gac cac ccc gtg gag ctg ttg gcc cga gac ttt gag aag aac 6369 Ala Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp Phe Glu Lys Asn 375 380 385 tac aat atg tac atc ttc cct gtc cac tgg cag ttt ggc cag ctg gat 6417 Tyr Asn Met Tyr Ile Phe Pro Val His Trp Gln Phe Gly Gln Leu Asp 390 c395 400 cag ac att gat ggg tac ctg tcc cac act gag ctg gcc cca ctg 6465 Gln His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu Ala Pro Leu 405 410 415 cgt gct ccc ctc atc ccc atg gaa cat tgc acc aca cgt ttc ttt gag 6513 Arg Ala Pro Leu Ile Pro Met Glu His Cys Thr Thr Arg Phe Phe Glu 420 425 430 435 acc tgt gac cta gac aac gac aag tac att gcc ctg gag gaa tgg gcc 6561 Thr Cys Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Glu Glu Trp Ala 440 445 450 ggc tgc ttt ggc atc aag gag cag gac atc aac aag gat ctg gtg atc 6609 Gly Cys Phe Gly Ile Lys Glu Gln Asp Ile Asn Lys Asp Leu Val Ile 455 460 465 taagttcacg cgtg cctg cctgaa ctctctccct ctgatgtgtc ccccctccca 6669 ttaccccctt gtttaaaatg tttggatggt tggctgttcc gcctggggat aaggtgctaa 6729 catagattta actgaataca ttaacggtgc taaaaaaaaa aaaaaaacaa ggtaagaaag 6789 aaactagaac ccaagtctca gcattttccc acataactct gaggccatgg cccatccaca 6849 gcctcctggt cccctgcact acccagtgtc tcactggctg tgttggaaac ggacttgtat 6909 aagctcaccg gccacaagca cgagatatct ctagctttca tttctgtttt gcatttgact 6969 cttaacactc acccagactc tgtgcttatt tcattttggg ggatgtgggc tttttcccct 7029 ggtggtttgg agttaggcag agggaagtta cagacacagg tacaaaattt gggtaaagat 7089 actgtgagac ctgaggaccc accagtcaga acccacatgg caagtcttag tagcctaggt 7149 caaggaaaga cagaataatc cagagctgtg gcacacatga cagactccca gcagcccggt 7209 cgactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa 7269 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa 7329 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggat 7375 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 1 <400> 3 ccgagagttc ccagcatcat <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 2 <400> 4 tcaaaccaat tcaccagtct

[0035]

[Sequence List Free Text] SEQ ID NO: 3 is primer 1
(Corresponding to the SPARC cDNA nucleotide sequence 72-91). SEQ ID NO: 4 shows the nucleotide sequence of primer 2 (corresponding to SPARC cDNA nucleotide sequence 1801-1820).

[0036]

Industrial Applicability According to the present invention, SPARC is expected to be used as a research reagent or pharmaceutical because it has various physiological activities such as angiogenesis, anti-cell adhesion activity, ECM reconstitution, and bone formation. Can be efficiently and inexpensively prepared in large quantities as a soluble fusion protein that does not lose its physiological activity and is easily prepared as a medicament.

[Brief description of the drawings]

FIG. 1 shows the entire amino acid sequence of the SPARC fusion protein.

[Explanation of symbols]

 A: Alanine (Ala) R: Arginine (Arg) N: Asparagine (Asn) D: Aspartic acid (Asp) C: Cysteine (Cys) Q: Glutamine (Gln) E: Glutamic acid (Glu) G: Glycine (Gly) H : Histidine (His) I: Isoleucine (Ile) L: Leucine (Leu) K: Lysine (Lys) M: Methionine (Met) F: Phenylalanine (Phe) P: Proline (Pro) S: Serine (Ser) T: Threonine (Thr) W: Tryptophan (Trp) Y: Tyrosine (Tyr) V: Valine (Val)

FIG. 2 shows an outline of construction of a pET32a (+)-SPARC vector.

FIG. 3 shows the results of gel electrophoresis (SDS-PAGE) of the purified sample.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ identification code FI C12P 21/02 C12P 21/02 C // A61K 38/00 A61K 37/02 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/09 C07K 14/195 C07K 14/435 C07K 14/47 C07K 19/00 C12P 21/02 A61K 38/17 CAPLUS (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. A protein represented by the following (a) or (b): (a) a protein having the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing; (b) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing , And a protein having the physiological activity of SPARC 2. A DNA encoding the protein according to claim 1. 3. The nucleotide sequence of SEQ ID NO: 2 in the sequence listing
DNA consisting of the nucleotide sequence from the 5209th position to the 6609th position. 4. A transformant obtained by introducing a recombinant vector into which a DNA according to claim 2 or 3 is inserted into a host cell, is cultured in a medium, and a protein encoded by the DNA is cultured in the culture. A method for producing a SPARC fusion protein, comprising producing and accumulating the protein, and collecting the protein from the culture.