JPH0731496A - Production of vascular permeability factor - Google Patents

Abstract

PURPOSE:To obtain the subject factor useful as a medicine inexpensively in a large amount in a short time by culturing a yeast transformed with a specific plasmid. CONSTITUTION:A yeast, for example, belonging to Saccharomyces is transformed with a plasmid expressing a vascular permeability factor which has also activity of promoting specific proliferation of vascular endothelial cells and the transformed yeast is cultured to produce the objective factor.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a vascular permeability factor produced in a trace amount by cells, and according to the present invention, isolation and purification conventionally required a great deal of time, labor and resources. The vascular permeability factor can be produced in a short time, in large quantities, and at low cost, and is widely used in the pharmaceutical and pharmaceutical industries.

[0002]

2. Description of the Related Art Vascular Permeability
Factor = VPF) or Vascular Endothelial Growth Factor (Vascular
A protein called Endothelial Growth Factor (VEGF) has the physiological activity of enhancing the permeability of substances in blood vessels and the activity of promoting the growth of vascular endothelial cells, which are the cells that make up the inner wall of blood vessels. (Ferrara, N. et al., Endocrine Reviews 13: 18-32,
1992). It has been proposed that vascular permeability factors may have clinically useful effects because they have these physiological activities (USP 4456550). Moreover, the present inventors have found that the factor is produced in a wide variety of tumors, and they are secreted into serum, and that they are useful as a so-called tumor diagnostic substance (Japanese Patent Application No. 36156).

This vascular permeability factor is an RNA that is transcribed from a single gene on the chromosome and is edited in various forms.
splicing), for example in the case of humans 4
It is believed that different lengths of protein are produced. Each protein has an extracellular secretory signal called a signal sequence on the amino terminal side, and is thereby transported to the outside of the cell. The protein transported to the outside of the cell has the signal sequence cleaved off, and the finally produced protein has four types of amino acid residues, 121, 165, 189, and 206, and their amino acid sequences are the same. (A short protein is a block protein in which some of the amino acids of the long protein are deleted in a block) (Japanese Patent Laid-Open No. 2-282398, Japanese Patent Publication No.
505705, special table 5-501350). It has been revealed that the synthesized protein is a dimer with two molecules bound by a disulfide bond, and that this dimer is the main body of a vascular permeability factor having biological activity (Ferrara, N. et al., Endo
crine Reviews 13: 18-32, 1992).

The vascular permeability factor has been separated and purified from the culture supernatant of various cultured cells. However, the method is complicated and the yield is extremely low. For example, in the case where cells derived from bovine pituitary vesicles were cultured and purified from the culture supernatant, 4 μg (Ferrara, N., et al., Methods in Enzymology 19) was finally obtained from 6 L of the supernatant.
8: 391-405, 1991), 1 L to 1 μg or less in the culture supernatant of guinea pig Line 10 tumor cells (Connoly, DT et al., J.
ournal of Clinical Investigation 84: 1470-1478, 198
9), 10 L to 5 μg in the supernatant of mouse Meth A tumor cells
(Clauss M. et al., J. of Experimental Medicine 172:
1535-1545, 1990), and only a very small amount was obtained.
In addition, in vitro culture of animal cells is a method that has problems in terms of economic efficiency and time efficiency such that the medium is expensive and that it takes a long time to grow the cells.

[0005]

Under the circumstances as described above, the present inventors have found that a physiologically active vascular permeability factor is
Research was conducted to establish a more economical and rapid manufacturing method.

[0006]

[Means for Solving the Problems] As a result of various studies, the present inventors left the secretory signal peptide of the gene (cDNA) of the active human vascular permeability factor as it was, or secreted it from the yeast α factor. The present invention was completed by finding that a dimer-forming active vascular permeability factor can be produced by replacing the signal with a prepro region containing a signal and introducing it into yeast, and culturing the yeast. That is, the present invention comprises the following four inventions. 1. An invention relating to a method for producing a vascular permeability factor, which comprises culturing and producing a yeast transformed with a plasmid that expresses a vascular permeability factor that also has a specific cell growth promoting activity on vascular endothelial cells. 2. An invention relating to a yeast Saccharomyces transformed with a plasmid expressing a vascular permeability factor which also has a specific cell growth promoting activity on vascular endothelial cells. 3. The invention relates to a DNA fragment in which the prepro region DNA of the yeast α factor and the coding region DNA of the vascular permeability factor which also has a specific cell growth promoting activity for vascular endothelial cells are linked in the same codon reading frame. 4. DNA fragment or vascular permeability factor in which the prepro region DNA of yeast α factor and the coding region DNA of the vascular permeability factor which also has a specific cell growth promoting activity for vascular endothelial cells are linked in the same codon reading frame Relating to yeast Saccharomyces transformed with a plasmid containing a coding region DNA fragment containing the signal peptide of E. coli.

[0007]

INDUSTRIAL APPLICABILITY According to the present invention, a vascular permeability factor can be rapidly and economically produced by yeast, which is much easier and more economical to culture than animal cells, and yeast culture has a long history. It is easy to scale up due to the large amount of experience and accumulation of facilities, and it will be possible to adapt to the production that meets the large industrial demand.

[0008]

EXAMPLES The present invention will now be described in detail based on examples, but the present invention is not limited to these. (a) Preparation of human vascular permeability gene-transferred yeast cDNA of human vascular permeability factor isolated from human promyelocytic leukemia cell HL60 was used as a commercially available yeast Saccharomyces cerevis
It was linked downstream of the Gal1 transcription promoter from siae. At that time, 2 which connect the entire coding region including the signal sequence of human vascular permeability factor (pIVPF) and which replace the signal sequence of human vascular permeability factor with the prepro region sequence of yeast factor α (pαVPF) Created a type. In Figure 1
The outline of the recombination process of pαVPF is shown. That is, yeast MAT
The restriction enzyme SacI recognition sequence was introduced between the prepro region of the α gene and the α factor peptide (upper row) to encode the signal peptide and mature protein of the human vascular permeability factor gene, that is, the coding region of the factor excluding the signal peptide. A restriction enzyme SacI recognition sequence was also introduced between the DNA coding region (middle row). The prepro region of MATα and the mature protein region of human vascular permeability factor are linked via the restriction enzyme SacI recognition sequence, and the gene is recombined so that the fusion protein is read at the correct genetic codon and cleaved by KEX2 endopeptidase. (Lower). These DNA fragments were ligated to a circular DNA containing a replication origin sequence derived from yeast 2μ plasmid, and commercially available yeast Saccharomyces cerevisiae INVSc2
Lithium acetate method (Lundblad, V. Current protocols: 13.
7.1.-13.7.2., 1989). pIVPF, and pαVPF
Yeast into which was introduced were isolated as YIVPF and YαVPF, respectively. Each yeast has the same characteristics as the parent yeast, except that it no longer requires uracil. Also, each transformed yeast was deposited at the Institute of Biotechnology, Institute of Biotechnology, AIST. Trust numbers are P-13731 and P-1373
It is 0.

(B) Secretion of human vascular permeability factor into the culture medium YC into which only YIVPF, YαVPF described above and circular DNA not containing human vascular permeability factor cDNA as a control were introduced
Were cultured under the following conditions. Yeast Nitrogen
en Base (YNB))-ammonium sulfate 2% -casamino acid 0.5% -histidine 80 mg / l was used as a basic medium, and raffinose 2% was used for preculture. 10 for each yeast
It was shake-cultured in 30 ml of the above preculture medium at 30 ° C. for 48 hours.
Next, 6 ml of the culture solution was inoculated into 600 ml of the same medium,
Culture was carried out with shaking at 48 ° C. for 48 hours. Yeast cells were collected from the culture solution at room temperature by centrifugation, and the collected cells were collected with YNB-
Ammonium sulfate 2% -casamino acid 0.5% -histidine 80 mg / l-
12 L of medium containing 5% galactose was inoculated again. This medium contains galactose and induces the expression of the human vascular permeability factor gene introduced downstream of the GAl1 transcription promoter. 12
The L medium was divided into four 3 L Erlenmeyer flasks and agitated and cultured at 30 ° C.

The state of cell growth of YIVPF yeast after re-inoculation
Secretion of human vascular permeability factor into the culture medium at D ₆₀₀ was measured by the enzyme immunoassay using the anti-human vascular permeability factor antibody described below, and the enzyme reaction of peroxidase was OD.
_The measurement was performed up to 6 days after re-inoculation with ₄₉₀ , and the results are shown in FIG. The number of cells reached about 22 OD 2 days after re-inoculation, and the density was maintained thereafter. On the other hand, the amount of human vascular permeability factor released in the medium reached the maximum value 2 days after re-inoculation,
After that, the same level was maintained until the 5th day, and it started decreasing on the 6th day. Therefore, the number of days for culturing is 2 after re-inoculation under the above conditions.
It was considered appropriate from the 5th to the 5th. Enzyme Immunoassay A separately prepared anti-VPF IgG was adsorbed on a 96-well plastic plate and coated with 2% bovineserum alubumin (BSA), which was used as a measurement plate. Put the measurement sample in each hole, leave it at room temperature for 2 hours, and then put the holes in phosphate buffered
Wash 6 times with saline (PBS). Add peroxidase-labeled antibody to this, leave it for 1 hour at room temperature, and then
Wash 9 times with PBS. After that, an enzyme substrate was added to each well to react with each other, and the color development was measured to measure VPF in the sample. In addition, the standard line is a rough VP obtained by calculating the number of units by another method.
F 2 was serially diluted and determined from the measured values in each ELISA.

(C) Confirmation of Human Vascular Permeability Factor Produced in Medium It was confirmed whether or not the human vascular permeability factor was contained in the culture solution of YIVPF, YαVPF, YC cultured under the same conditions. Each culture supernatant was serially diluted to 10 μL-0.001.
FIG. 3 shows the results of examination using the above-mentioned enzyme-linked immunosorbent assay for human vascular permeability factors up to μL. As a result YαV
The culture supernatant of PF contained about 10 times as much substance as the culture supernatant of YIVPF that reacts with the anti-human vascular permeability factor antibody. On the other hand, no reactive substance was detected in the control YC culture supernatant,
It was shown that the antibody-reactive substances found in the culture supernatants of YIVPF and YαVPF are derived from the introduced human vascular permeability factor gene.

The vascular permeability factor has been shown to be a dimer of two polypeptides formed by a disulfide bond (Ferrara, N. et al., Endocrine Revi.
ews 13: 18-32, 1992). Therefore, if the human vascular permeability factor produced by yeast cells also does not form a dimer, it cannot be expected to function as a vascular permeability factor that retains its activity. Therefore, in order to add consideration to this point, YIVPF, YαVPF, Y
Add twice the amount of acetone to the culture supernatant of C, collect the insolubilized components by centrifugation, and perform SDS polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol, which is a disulfide bond reducing agent. FIG. 4 shows the results of analysis of substances produced by the so-called Western blot technique, in which the sample was transferred from the gel to a nylon membrane after electrophoresis and detected with an anti-human vascular permeability factor antibody. Nothing was detected in the control YC (lanes 1 and 4). In YIVPF, a molecular weight marker was detected between 30 and 46 kilodaltons under non-reducing conditions (lane 2) and a molecular weight marker between 14 and 20 kilodaltons under reducing conditions (lane 5). In addition, in YαVPF, molecular weight marker 3 under non-reducing conditions
It was detected both between 0 and 46 kilodaltons and between the molecular weight markers 14 and 20 kilodaltons (lane 3) and under reducing conditions between the molecular weight markers 14 and 20 kilodaltons (lane 6). It was

The above results are summarized as follows. It was shown that both extracellular secretory signals of YIVPF, which is a human vascular permeability factor, and YαVPF, which is derived from yeast α factor, secrete human vascular permeability factor derived from the introduced gene into the medium. It was suggested that YαVPF was about 10 times higher in the amount secreted into the medium than YIVPF. The human vascular permeability factor produced by YIVPF is completely 2
While it was suggested that they form a monomer, YαV
It was suggested that in PF, the dimer-forming one and the non-dimer-forming one were mixed in the medium.

The above findings are noteworthy as the findings suggesting that the amount of substances produced and the manner of production are influenced by the influence of the added extracellular secretion signal. It has been reported that the prepro region of α factor is cleaved by an endopeptidase called KEX2 (J
ulius, D. et al., Cell 37: 1075-1089, 1984), which is different from mere extracellular transfer of secretory proteins. It is expected that such differences in the mechanisms involved in extracellular secretion will influence the amount of substances produced and the state of aggregation of molecules.

(D) Purification of human vascular permeability factor from culture medium YIVPF or YαVPF was cultured under the above conditions, and human vascular permeability factor was purified from the culture supernatant by the following method. Yeast was removed from 12 L of the cultivated culture solution by centrifugation,
Furthermore, the supernatant was passed through a 0.45 μm filter to remove suspended matter. Next, the sample was finally concentrated from 12 L to 50 mL using an ultrafiltration membrane with a molecular weight cut off of 10,000 (PLGC10000 manufactured by Nippon Millipore Limited). 1 concentrated sample
It was dialyzed against 0 mM acetate buffer (pH 5.2) -0.1 M sodium chloride.

The dialyzed sample was subjected to (strong) cation exchange column chromatography. A cation exchange chromatography column (TSK-SP650 manufactured by Tosoh Corporation) was preliminarily equilibrated with 10 mM acetate buffer (pH5.2) -0.1 M sodium chloride, and the sample after dialysis was flown. The components not adsorbed on the column were thoroughly washed with 10 mM acetate buffer (pH5.2) -0.1M sodium chloride, and then the sodium chloride concentration was 0.1M-
2.1 mM linear gradient with 10 mM acetate buffer (pH
In 5.2), the substance adsorbed on the column was eluted. The eluate was fractionated with a fraction collector, and the human vascular permeability factor contained in each fraction was examined by the enzyme immunoassay described above.

Ammonium sulfate was added to the fractions which became positive by the enzyme immunoassay, and ammonium sulfate precipitation was carried out under a saturated condition to collect a sample.
The precipitate was redissolved in 0.2 ml of 10 mM acetate buffer (pH 5.2) -0.1 M sodium chloride and subjected to gel filtration column chromatography. Gel filtration column (Pharmacia Superdex-7
5) was equilibrated with 10 mM acetate buffer (pH5.2) -0.1 M sodium chloride in advance, and the redissolved sample was applied to the column. The solution was passed through the column at a constant rate, the eluate was fractionated by a fraction collector, and the human vascular permeability factor contained in each fraction was examined by the enzyme immunoassay described above.

A part of the fractions which became positive by the enzyme immunoassay was taken and analyzed by SDS15% polyacrylamide gel electrophoresis according to the method of Laemley (Laemmli, UK, Nature 2).
27: 680-685, 1970). That is, for each sample prepared from the culture supernatant of YIVPF, YαVPF, in the presence of 2-mercaptoethanol, which is a disulfide bond reducing agent (lane 1: YIVPF, 2: YαVPF), or in the absence thereof ( Lane 4: YIVPF, 5: YαVPF) After the electrophoresis and the gel was stained with silver, the results are shown in FIG. Both YIVPF and YαVPF samples have a single band between 30 and 46 kilodaltons under non-reducing conditions and a single band between 14 and 20 kilodaltons under reducing conditions. Was observed. From this, it was shown that the human vascular permeability factor forming a dimer with a disulfide bond was purified.

(E) Activity of Human Vascular Permeability Factor Whether the purified substance retains its physiological activity as a human vascular permeability factor is determined by its proliferation against vascular endothelial cells, which is one of the physiological activities of vascular permeability factor. Confirmed by promoting activity
(Ferrara, N., and Henzel, WJ, Biochem.Biophys.Res.Co
mmun. 161: 851,1989). The physiological activity of cultured human vascular endothelial cells was confirmed as follows. Human umbilical cord-derived vascular endothelial cells to a culture plate 12 well (HUVEC, Kurabo) 10 ⁴
Sow, assay medium (RPMI1640 45%-DMEM 45%-fetal bovine serum 10%-bovine insulin 10 mg / l-human transferrin 5 mg / l-0.01 mM 2-mercaptoethanol
-0.01 mM 2-aminoethanol-10 nM sodium selenite) 1 mL of various amounts (0-125 μl) of purified yeast-produced human vascular permeability factor solution was added, and the temperature at 37 ° C 5% 2
The results of comparing the number of cells after 5 days of culturing under the carbon oxide condition are shown in FIG. Both YIVPF (A) and YαVPF (B) promoted cell growth in a dose-dependent manner. From this result, it was shown that the purified yeast-produced human vascular permeability factor retained physiological activity.

[0020]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, in the production of vascular permeability factor, the substance, which was conventionally only purified in a very small amount from the culture supernatant of animal cells, was activated with yeast. It shows a method for producing the protein in a retained form in a medium and purifying it. As a result, the following effects can be expected.

1. In the conventional method, since the raw material was obtained by culturing animal cells, a large amount of cost was economically required to obtain a small amount of the purified product. Now that it can be produced, it has become much easier and cheaper to manufacture.

2. In addition, since yeast cells are much more active in growth than animal cells, it is possible to achieve the purpose in a very short period of time compared with the conventional method of culturing cells and procuring raw materials. Is now possible.

3. Yeast culture has a long history of industrial application, and its accumulated technology and knowledge make it easy to apply the technology to an industrial scale. Therefore, using this method, it is possible to easily meet the industrial demand that will occur in the future.

[Brief description of drawings]

FIG. 1 is a diagram showing an outline of gene recombination of an extracellular secretion signal of a vascular permeability factor.

FIG. 2 is a diagram showing cell growth of recombinant yeast YIVPF and production of vascular permeability factor in the medium. In the figure, ◯ indicates the amount of yeast cell growth, and □ indicates the amount of vascular permeability factor production.

FIG. 3 is a diagram in which the amount of vascular permeability factor produced in a medium was measured by enzyme immunoassay with serial dilution. □ indicates YC, Δ indicates YIVPF, and circle indicates YαVPF.

FIG. 4 is a diagram showing that the culture solution was subjected to acetone precipitation, and the sample was analyzed by Western blotting in the presence (+) of 2-mercaptoethanol and in the absence (−) of 2-mercaptoethanol. -), Lane 2: YIVPF (-), Lane 3: YαV
PF (−), lane 4: YC (+), lane 5: YIVPF (+), lane 6: YαVPF (+).

[FIG. 5] A diagram of vascular permeability factors purified from YIVPF and YαVPF culture supernatants, analyzed by SDS polyacrylamide gel electrophoresis, showing that lanes 1 (YIVPF) and 2 (YαVPF) were in the absence of 2-mercaptoethanol. , Lane 4 (YIVPF), 5 (YαVP
F) was treated in the presence and lane 3 is a molecular weight marker.

[Fig. 6] Fig. 6 is a diagram showing the growth promoting effect of vascular permeability factor purified from YIVPF and YαVPF culture supernatants on vascular endothelial cells. (A) is a sample purified from YIVPF, (B) is purified from YαVPF. It is the figure which tested with the sample.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1: 865) (C12N 1/19 C12R 1: 865) (72) Inventor Iwao Omori Tsukuba, Ibaraki Prefecture No.2 Okubo, Tokushima Synthetic Chemical Industry Co., Ltd. Tsukuba Research Center

Claims (4)

[Claims] 1. A method for producing a vascular permeability factor, which comprises culturing and producing yeast transformed with a plasmid expressing a vascular permeability factor also having a specific cell growth promoting activity on vascular endothelial cells. 2. A yeast Saccharomyces transformed with a plasmid expressing a vascular permeability factor which also has a specific cell growth promoting activity on vascular endothelial cells. 3. A DNA fragment in which the prepro region DNA of yeast α factor and the coding region DNA not containing the signal peptide of the vascular permeability factor which also has a specific cell growth promoting activity for vascular endothelial cells are linked in the same codon reading frame. . 4. A yeast Saccharomyces transformed with a plasmid containing the DNA fragment of claim 3 or a coding region DNA fragment containing a signal peptide of a vascular permeability factor which also has a specific cell growth promoting activity for vascular endothelial cells.