JP3062841B2 - Polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel polypeptide secreted by cultured cells established from mammalian liver cancer tissue, and can be used in the field of drugs for liver diseases and the like, and reagents for clinical tests thereof. It is.

[0002]

2. Description of the Related Art HuH-established from human liver cancer tissue
7 hepatoma cell lines are known to grow in serum-free media and secrete various serum proteins (Nakabayashi et al., Cancer Research, 1982, 42,
3858 to 3863).

[0003] It has been reported that a basic fibroblast growth factor (FGF) was isolated and purified as a protein secreted from hepatoma cells. This protein binds strongly to heparin-sepharose and is first eluted with aqueous sodium chloride at concentrations of 1 mol / l or more (Kl
agsbrun et al., Proceeding national academic sci.
ence, 1987, 84, 1839-1843.
page).

It has been reported by Mckeehan that acidic FGF promotes the proliferation of HuH-7 cells.

[0005] Further, cell culture factors partially purified from the culture supernatant of human hepatoma cultured HuH-7 cell line through ion exchange chromatography, affinity chromatography using heparin as a ligand and gel filtration chromatography. (H. Nakamura et al., Clinica Simica Acta)
a Chimica Acta) 1989, 183, 273-28
4).

[0006]

However, a further purified product of the above-mentioned cell factor derived from liver cancer has not yet been obtained, and its structure has not been elucidated.

An object of the present invention is to provide a purified product of a cell growth activating factor derived from human liver cancer, thereby contributing to its practical use in the field of drugs for liver diseases and the like, or clinical test reagents thereof.

[0008]

The polypeptide according to the present invention has a molecular weight in the range of 20,000 to 35,000, and 20 residues from the N-terminus are any of the following (1), (2) and (3): Is composed of the amino acid sequence of

Sequence (1) Ser Asn Asn Gln Lys Glu Tyr Lys Cys Gly Asp Leu Val Phe Ala Lys Met Lys Gly Tyr Sequence (2) Ser Asn Asn Gln Lys Glu Tyr Lys Ser Gly Asp Leu Val Phe Ala Lys Met Lys Gly Tyr sequence (3) Ser Asn Asn Gln Lys Glu Tyr Lys Thr Gly Asp Leu Val Phe Ala Lys Met Lys Gly Tyr

The polypeptide of the present invention having the above amino acid sequence has cell proliferation activity on Swiss 3T3 cells,
The activity is deactivated by any of the following treatments (a), (b), (c) and (d).

(A) heat treatment , eg, 100 ° C. for 5 minutes; (b) treatment with a reducing reagent , eg, treatment with a reducing reagent such as dithiothreitol; (c) treatment with an acid solution , eg, treatment with a 1 mol / liter acetic acid solution (d ) Trypsin aqueous solution treatment , for example, 0.5 mg / milliliter trypsin aqueous solution treatment

The polypeptide of the present invention having the above-mentioned amino acid sequence can be obtained from a culture supernatant of human hepatoma cell line HuH-7 by ion-exchange chromatography, affinity chromatography using heparin as a ligand, gel filtration chromatography and reverse phase chromatography. It is produced by successively passing through each purification step of chromatography.

The polypeptide according to the present invention is derived from human liver cancer and has the following cell proliferation activity.
Hereinafter, it is referred to as a human liver cancer-derived growth factor, and is abbreviated as HuHGF. In order to produce the polypeptide according to the present invention, first, ion exchange chromatography, affinity chromatography using heparin as a ligand and gel filtration chromatography are performed from the culture supernatant of HuH-7 hepatoma cells established from human hepatoma tissues. Was used to purify HuHGF up to about 1000-fold. Cell proliferation activity is 3T in Switzerland
Assayed in 3 cells. HuHGF after purification in this manner
Promoted the growth of Swiss 3T3 cells at a concentration of 1-2 μg / milliliter. HuHGF was heat and acid labile and lost proliferative activity upon trypsinization.
HuHGF was completely inactivated under reducing conditions, suggesting that a disulfide bond is required to maintain activity.

After the gel filtration chromatography, several protein bands were observed by polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate.
Further purification was performed using reverse phase chromatography.
As a result, a single band was obtained by the above electrophoresis, and the molecular weight was estimated to be between 20,000 and 35,000. These results
HuHGF is epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDG
F), suggesting that it is a growth factor different from tumor growth factor (TGF) and the like. However, HuHGF has an affinity for heparin.

The elution behavior of HuHGF such as chromatography will be described. Klagsbrun et al. Reported that basic FGF was isolated and purified from hepatoma cells as described above, and that this protein strongly bound to heparin-sepharose and was eluted for the first time in an aqueous solution of sodium chloride having a concentration of 1 mol / liter or more. doing. On the other hand, HuHGF is 0.1.
Desorption from heparin-Sepharose with 6-0.7 mol / l aqueous sodium chloride solution. From these results, H
It can be seen that uHGF is different from basic FGF. HuHG
The isoelectric point of F is on the acidic side, at which point it is the acidic FG
Similar to F.

As described above, Mckeehan shows that acidic FGF is Hu
It reports that it promotes the growth of H-7 cells, suggesting that HuHGF is similar to acidic FGF. However, the molecular weight of acidic FGF is about 17,000 and heparin binding is slightly stronger than HuHGF.

When the amino acid sequence of a single band purified by reverse phase chromatography was analyzed, the amino acid sequence of the 20 residues from the N-terminus did not match the sequence of the polypeptide reported so far. That is, HuHGF is a novel polypeptide.

Some of the growth factors are used for clinical diagnosis and treatment. For example, EGF is expected as a therapeutic agent for burns and a therapeutic agent for corneal damage. It has been reported that FGF activity is increased in urine of patients with bladder cancer and kidney cancer (Ch
odak et al., Cancer research, 1
988, 48, 2083-2088). PDG
F is under development for cartilage damage treatment and wound healing. T
GF-alpha is often observed in body fluids of cancer patients,
There are reports that the strength of its activity correlates with patient survival. (Arteaga et al., Cancer Research
search), 1988, 48, 5023 pages-5028
page). Human erythropoietin is known as a hematopoietic hormone as a diagnostic agent for anemia and a therapeutic agent for anemia by renal dialysis. Granulocyte-macrophage colony-stimulating factor is expected as a therapeutic drug for side effects of cancer chemotherapy and radiation therapy and for granulocytopenia associated with hyperthyroidism.

Similarly, the polypeptide HuH according to the invention
GF also has the potential to be useful for various diagnoses and treatments, including liver disease. Hepatocyte growth factor is known as a hepatocyte growth factor, which is also expected as a diagnostic agent for non-A non-B hepatitis and the like and a therapeutic agent for hepatocyte regeneration. Is also expected to be applied in this area.

[0020]

【Example】

1. Purification of HuHGF (1) Preparation of culture supernatant HuH-7 cells (JCRB0403, Cancer Research Foundation Cell Bank) were heated at 37 ° C. in a carbon dioxide incubator using a 75 cm ² flat bottom flask for cell culture.
The cells were cultured in an atmosphere at a temperature of 5 ° C. and a CO ₂ content of 5%. The culture solution used is as shown in Table 1 below.

[0021]

[Table 1]

The culture solution was replaced every two days, and the culture supernatant was collected at the time of replacement, and the temperature was kept at -3 until the subsequent purification.
Stored at 0 ° C. Subculture is trypsin-EDT
A was performed by treating the cells with A.

(2) Purification of HuHGF by Chromatography 5 liters of the preserved culture supernatant was passed through an ultrafiltration device (YM-5).
The solution was concentrated to 300 ml using a membrane interior (manufactured by Amicon). The concentrated solution was dialyzed against 50 mmol / liter of tris (hydroxymethyl) aminomethane hydrochloride buffer (set to pH 8.0; this buffer is hereinafter abbreviated as Tris buffer). A cellophane tube (fraction molecular weight 12,000 to 14,000, manufactured by Union Carbide) was used as the semipermeable membrane.

I) Ion exchange chromatography After dialysis, DEAE-Sepharose C was used as an ion exchanger.
The dialysate was subjected to ion exchange chromatography using a column (1.6 × 50 cm, manufactured by Pharmacia) packed with L-6B. That is, after applying the concentrated solution to the column, the column was sufficiently washed with 450 ml of Tris buffer, and the adsorbate was eluted with Tris buffer containing sodium chloride. As an elution method, a linear gradient elution was performed in a sodium chloride concentration range of 0 to 0.6 mol / liter. The eluate was collected in fractions of 2 ml each, and 120 ml fractions showing proliferative activity on Swiss 3T3 cells were combined and used for the next affinity chromatography step.

Ii) Peparin affinity chromatography The combined active fractions were dialyzed against 2,000 milliliters of Tris buffer using the semipermeable membrane. The obtained dialysate was applied to a column (1.3 × 3.8 cm, manufactured by Pharmacia) packed with heparin-Sepharose CL-6B, and the column was washed with 360 ml of Tris buffer. Elution was performed with a linear gradient of Tris buffer containing .about.1.5 mol / l sodium chloride. The eluate was collected in 2 ml fractions, and 100 ml fractions showing proliferative activity on Swiss 3T3 cells were combined and used for the next gel filtration chromatography step.

Iii) Gel Filtration Chromatography The combined active fractions were dialyzed against 2,000 milliliters of Tris buffer using the semipermeable membrane. The obtained dialysate was applied to a column (2.6 × 100 cm, manufactured by Pharmacia) packed with Sephadex G-150, and gel filtration chromatography was performed. The eluate was collected in fractions of 2 ml each, and the 60 ml fractions having a molecular weight of 50,000 or less and showing proliferative activity on Swiss 3T3 cells were combined and used for the next reverse phase chromatography step.

IV) Reversed phase chromatography The combined fractions were separated by ultrafiltration as described above.
Concentrated by a factor of 1 and the concentrate was subjected to reverse phase chromatography. The reverse phase chromatography conditions are as follows. The column used was a silica-based reversed-phase column (Cosmosil 5C-18, 4.6 × 150 mm, manufactured by Hanui Tesque).
The elution was performed with a linear gradient of acetonitrile at a concentration of 0 to 50% by weight. However, 0 to 30% by weight in the first 10 minutes, 30 to 50% by weight in the next 60 minutes
The ratio of acetonitrile was increased in two steps. Water containing 0.1% by weight of trifluoroacetic acid was used as the base solution. This chromatography was performed at a flow rate of 1 ml / min, and fractionation was performed in 1 ml portions.

The fractions thus obtained were subjected to SDS polyacrylamide gel electrophoresis. As a result, a single protein band was observed at a molecular weight of about 30,000. The 22nd fraction with high proliferative activity on Swiss 3T3 cells was used for the following amino acid sequence analysis.

[0029] 2. Measurement of Cell Proliferating Activity on Swiss 3T3 Cells The cultured cells used for the measurement were Swiss 3T3 fibroblasts provided by Dr. Kitano of Osaka University School of Medicine.

The above activity was determined by measuring the ability to proliferate Swiss 3T3 cells. That is, Swiss 3T3 cells were placed in a 96-well microplate at 3 × 1 per well.
0 ³ cells with DME culture 200μ liters (Flow Laboratories, Inc., 10% by volume fetal calf serum (FCS)
) Was seeded. Incubate for 48 hours in a carbon dioxide gas incubator (CO ₂ : 5%, temperature 37)
° C), and each well was replaced with 200 µl of a DME culture solution containing FCS at a concentration of 0.2% by volume, and the culture was continued for further 36 hours. In this state, the cells were stimulated with the culture supernatant or FCS as a positive control and phosphate buffer as a negative control in order to examine the cell growth activity of the HuH-7 culture supernatant on Swiss 3T3 cells. Eighteen hours later, 0.5 μCurie of ³ H-thymidine (manufactured by Amersham) was added to each well, and the above-mentioned thymidine was added to the DNA for 37 hours.
℃ In was taken 6 hours, were then incorporated into DNA ³
The radioactivity of H-thymidine was measured.

The radioactivity incorporated into Swiss 3T3 cells for the stimulated samples is shown in Table 2 below.

[0032]

[Table 2]

* Indicates the amount of protein per milliliter of culture supernatant. The amount of this protein is determined by the ultraviolet absorption method (UV280n
m).

The cell proliferation activity in each of the above purification steps was also measured in the same manner as described above.

[0035] 3. Changes in the activity of HuHGF by various treatments In order to clarify the properties of HuHGF, it was examined how the cell growth activity on Swiss 3T3 cells was changed when the HuHGF was treated with the following reagents. The HuHGF used was a sample purified by the above reverse phase chromatography. Assuming that the activity of this sample was 100%, the remaining activities after various reagent treatments are shown in Table 3 below.

[0036]

[Table 3]

4. Amino acid sequence analysis Purified HuHGF according to the present invention was applied to a gas-phase sequencer, and amino acid sequence analysis was performed for 20 cycles. Applied Biosystems Model 477A was used as a sequencer, and Applied Biosystems
A 120A type was used. As a result, it was found that 20 residues from the N-terminal consisted of any one of the amino acid sequences of SEQ ID NOs: (1), (2) and (3) shown in the following sequence listing.

This amino acid sequence is completely novel.

[0039]

According to the present invention, it is possible to provide a purified product of a cell growth activating factor derived from human liver cancer, and to commercialize it in the field of drugs for liver cancer diseases and others, or clinical test reagents thereof. Can contribute to.

[0040]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment: N-terminal fragment Origin: Human hepatoma cultured cell Direct origin: JCRBO403, Cancer Research Foundation Cell bank SEQ ID NO: 2 Sequence length: 20 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment: N-terminal fragment Origin: Human hepatoma cultured cell Direct origin: JCRBO403, Cancer Research Foundation Cell bank SEQ ID NO: 3 Sequence length: 20 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment: N-terminal fragment Origin: Human hepatoma cultured cell Direct origin: JCRBO403, Cancer Research Foundation Cell bank

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // C12P 21/02 C12P 21/02 A (C12P 21/02 C12R 1:91) (72) Inventor Shuji Nakamura Suita-shi, Osaka 4-2 Senrioka Naka (72) Inventor Yasushi Kobe 20-16 Midorigaoka, Ashiya-shi, Hyogo (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 14/47 C12P 21/00-21/02 BIOSIS ( DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. Culture of human hepatoma cultured HuH-7 cell line
Ion exchange chromatography and heparin from the culture supernatant
Affinity chromatography as a ligand, gel
Filtration chromatography and reverse phase chromatography
Polypeptide obtained through each of the purification steps
Has a molecular weight in the range of 20,000 to 35,000, and 20
The residue is composed of any one of the following amino acid sequences (1), (2) and (3), and the sequence (1) Ser Asn Asn Gln Lys Glu Tyr Lys Cys Gly Asp Leu Val Phe Ala Lys Met Lys Gly Tyr sequence (2 Ser Asn Asn Gln Lys Glu Tyr Lys Ser Gly Asp Leu Val Phe Ala Lys Met Lys Gly Tyr Sequence (3) Ser Asn Asn Gln Lys Glu Tyr Lys Thr Gly Asp Leu Val Phe Ala Lys Met Lys Gly Tyr Swiss 3T3 cells A polypeptide which has a cell growth activity and is inactivated by any of the following treatments: (a) heat treatment (b ) treatment with a reducing reagent (c) treatment with an acid solution (d) treatment with an aqueous trypsin solution .