JPH0453489A - Genetic engineering production of prourokinase - Google Patents

Abstract

PURPOSE:To produce the subject new enzyme in high efficiency in a mass by culturing a transformant transformed with a manifestation vector containing a cDNA coding a prourokinase originated from endothelial cell of human vessel. CONSTITUTION:A host cell is transformed with a manifestation vector containing a cDNA coding a prourokinase originated from endothelial cell of human vessel and the obtained transformant is cultured to produce prourokinase. The cDNA coding the prourokinase can be produced by conventional process from whole mRNA extracted from the subcultured cell of the endothelial cell of human umbilical cord vein.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for producing prourokinase using genetic engineering. More specifically, a genetic engineering method for producing prourokinase by transforming a host cell with an expression vector containing a cDNA encoding prourokinase derived from human vascular endothelial cells and culturing the resulting transformant;
The present invention also relates to human vascular endothelial cell-derived cell lines that can be used as host cells for expressing recombinant DNA.

<Prior Art> Plasminogen activator and its precursors, which can be obtained from human urine, human cells, etc., are known as thrombolytic agents effective in treating myocardial infarction, cerebral infarction, etc. Thus, for example, urokinase prepared from human urine (Williams J.

R, B,, Br, J, EXD, Pat
hol,,32. 530), prourokinase produced from human kidney cells or cancer cells [T, Suyagi
a et at,, J, Biol, Chew26
0 (22), 12377 (1985): DC3tu
mp et al., J. Biol, CheIl.
l,, 261 (3).

1274 (1986)] or tissue plasminogen>-7 activator [0,C, Rijken et al.
,, J. Biol, Chet, 256 (13
) (1981)].

Urokinase and tissue plasminogen activator require large doses when used clinically, and it has been pointed out that they have the side effect of decomposing fibrinogen in the circulation, which tends to lead to bleeding. .

On the other hand, prourokinase does not degrade fibrinogen in the blood, and has a strong affinity for fibrin, so it exerts its effect only on blood clots. Furthermore,
Less likely to be inhibited by plasminogen activator inhibitors in the blood. Therefore, prourokinase is expected to be a thrombolytic agent that does not show bleeding tendency.
Its development is actively underway at home and abroad.

On the other hand, as mentioned above, conventional human plasminogen activators such as prourokinase or their precursors are derived from human urine, human kidney IB cells or human cancer cells. In contrast, since toplasminogen activator originally acts within blood vessels, there has been a need for plasminogen activator or its precursor derived from human vascular endothelial cells.

Regarding the conventional production method of plasminogen activator or its precursor, it is difficult to collect materials, and nutritional conditions must be adjusted to increase the growth efficiency of cells producing plasminogen activator or its precursor. It is necessary to use a suitable culture medium, a large amount of contaminant proteins are produced, and there are many problems when performing isolation and purification (and it is difficult to mass produce).

<Problems to be Solved by the Invention> An object of the present invention is to provide a genetic engineering production method that enables mass production of human vascular endothelial cell-derived prourokinase, which is a new type of prourokinase.

Another object of the present invention is to provide a host cell for expressing recombinant DNA comprising an established cell line derived from human vascular endothelial cells.

<Means for Solving the Problems> As a result of intensive exploration of cultures of human vascular endothelial cells, the present inventors discovered that a new type of pro-urokinase could be obtained, and then cloned a cDNA encoding this pro-urokinase. succeeded in. This CDN
Recombining a new type of prourokinase using A
The present invention was completed by establishing a method for mass production using NA technology and by discovering that established cell lines derived from human vascular endothelial cells can be widely used as host cells for expressing recombinant DNA.

The present invention provides a method for producing prourokinase, which comprises transforming a host cell with an expression vector containing a cDNA encoding prourokinase derived from human vascular endothelial cells, and culturing the resulting transformant; and The present invention relates to a host cell for expressing recombinant DNA consisting of an established cell line derived from human vascular endothelial cells.

The pro-urokinase targeted by the present invention is pro-urokinase derived from human vascular endothelial cells, and can be obtained from the culture supernatant of a cultured cell line of human vascular endothelial cells obtained from, for example, human dandelion veins. It is something.

A cDNA encoding prourokinase derived from human vascular endothelial cells can be obtained, for example, as follows. Vascular endothelial cells are obtained from the human sea bream vein, and total mRNA is extracted from the subcultured cells.

Adsorb the total mRNA onto an oligo dT cellulose column,
Next, the mRNA is eluted, and the base sequence of the cDNA of known human wise cell-derived prourokinase [8, N
agai et at, Gene, 36, 183
-188 (1985)] is used as a probe, and mRNA that hybridizes with this probe is collected. Using this mRNA, the method of Gubler et al. [0, Gubler et al.
al., Gene, 25.263 (1983
)] to create a CDNA library, and use the above probe to create a CDNA library that encodes the target pro-urokinase.
Isolate the DNA-containing link. By extracting cDNA from this clone, a CD encoding prourokinase derived from human vascular endothelial cells used in the present invention was obtained.
NA can be obtained. The base sequence of the CDNA encoding prourokinase derived from vascular endothelial cells obtained from human zona veins is shown in FIG. FIG. 1 also shows the amino acid sequence determined from the base sequence. In the amino acid sequence of prourokinase shown in Figure 1, the amino acid residue at position 12) from the N-terminus is leucine; The amino acid residue at position 12) of the known prourokinase is proline [8. Nagai et al., Gene, 36
．． 183-188 (1985) 1illiai E,
Holmes et al., 8+.

/Techology, Vol. 3. Oct.9
23-929 (1985) Nijierde, P6. e
tal, Proc, MatAcad, Sci
, USA, 81.4727 (1981): Ri
ccio A, et at, Nucleic
Ac1dsRe5earch.

Eyu (8), 2759 (1985)]. 19 in Figure 1
The amino acid residue at position 4 is isoleucine, whereas the corresponding amino acid residue in prourokinase derived from human lineoblasts and human liver is methionine.

Regarding the cDNA base sequence of prourokinase shown in Figure 1, the codons corresponding to amino acid residues at positions 12) and 313 are different from the cDNA base sequence encoding prourokinase derived from human kidney cells. 12)
．． Codons corresponding to amino acid residues at positions 194.313.340.345 and 346 are different from the cDNA base sequence encoding prourokinase derived from human lineoblasts. Also, 12). Codons corresponding to amino acid residues at positions 340, 345 and 346 are different from the cDNA jm base sequence encoding prourokinase derived from human pharyngeal cancer cells. In addition, the codons for amino acid residues at positions 12) and 194 are different from the cDNΔ base sequence encoding human liver-derived prourokinase.

The base sequence of CDNA used in the present invention is not limited to that shown in FIG.

Even if it is a cDNA base sequence variant that is substantially equivalent to the cDNA base sequence shown in Figure 1, that is, based on degeneracy of the genetic code, deletion or addition of the base sequence, etc. A cDNA encoding a polypeptide having physiologically equivalent properties to prourokinase represented by the amino acid sequence shown can also be used in the same manner as the cDNA shown in FIG.

The expression vector used to introduce the cDNA encoding human vascular endothelial cell-derived prourokinase of the present invention may be any expression vector and is not particularly limited. For example, when using E. coli as a host cell, 1) BR322 [Bol
ivar, F. Gene, 2゜95 (1977
)], pvcs [Vierira Jand M
essing, J., Gene 9.259
(1982)], pKK223-3 [deBo
er, H, AProc, Natl, Acad, S
ci, USA, 78.2) (1983)]. When using Bacillus subtilis as a host cell, for example, pUBllo [eggins, H, .
LOVett, P., S., & Duval.
l EJ, Proc, Natl, Aca
d, Sci, USA, 75.1423 (1978
)], pc194 [Byeon, y.

J. Bac., 158.543-550 (1984)
] etc. When using animal cells as host cells, examples include SV40 and bovine papillomavirus, and DSVL [
Te1pleton, D et al, HQl,
Ce1lBio1. ．． 4, 817 (1984)]
, pB PV [5arver, Net al,,
Mo1ecular and Cl1nical As
pectsp, 515. Howley, P.H.
, and Broker T, R, eds.].

c encoding prourokinase in these vectors.
DNA can be introduced by conventional methods using appropriate restriction enzyme sites. When using animal cells as host II cells, the expression vector contains a DNA sequence encoding dihydrofolate reductase (dhfr) protein in order to amplify the copy number of the expression vector and increase the expression level of prourokinase. It's okay.

A DNA sequence encoding the dhfr protein component can be obtained, for example, from a plasmid such as DsV2-dhfr (ATCC 37146). Also, JP-A-1-2851
A cDNA encoding pro1 crokinase may be introduced into a plasmid vector containing DNA sequences encoding factor L and dhfr protein described in Japanese Patent No. 90.

In addition to the above-mentioned DNA sequence, the expression vector contains a promoter sequence, a Shine-Dalgarno sequence, a sequence encoding a signal peptide, etc. necessary for expressing the target protein. These DNA
The sequence is known and can be arbitrarily selected depending on the host-vector system used.

The host cell used in the present invention may be any one and is not particularly limited, and can be arbitrarily selected depending on the type of expression vector. For example, when pBR322 is used as a vector, E. coli HB 101 or the like can be used as a host cell. When using a vector for animal cells, Chinese hamster (CHO) cells, BHK cells, Namalva cells, etc. can be used. When the expression vector contains a DNA sequence encoding the dhfr protein, for example, dhfr protein derived from CHO-Kl cells
fr protein gene-deficient cells [LlrlabG, a
nd Chasin L, Proc, Natl.
Acad, 5ciUSA, 7yu, 42)6 (19
80) etc. are used. In this case, it is of course possible to use a single CHO cell which naturally contains the dhfr protein gene.

The prourokinase targeted by the present invention is derived from human vascular endothelial cells, and therefore, it is suitable to use human vascular endothelial cell-derived cell lines as host cells. This established cell line can be obtained by, for example, enzymatic detachment from human ligament veins [JaNee, E., AJ, Clnical
Investigation, Vol, 52, 27
45 (1973)], and then subculturing the cells repeatedly. As the established cell line, a human vascular endothelial cell-derived cell line that can be cultured in a serum-free medium is particularly suitable, and by using such an established cell line, an industrially advantageous production method for prourokinase can be achieved. .

Established cell lines that can be cultured in a serum-free medium are obtained by the method described above, and are subcultured repeatedly in a medium with gradually decreasing serum concentration. It can be prepared by obtaining cells that can be expanded in a basal medium containing.

The human vascular endothelial cell-derived cell line thus prepared is
In addition to expressing prourokinase, it can be widely used to express other common useful proteins. Since this established cell line is derived from normal human cells, it is possible to avoid contamination of harmful proteins with proteins manufactured using this cell line as a host am, and to obtain proteins with low antigenicity to humans. is possible and has many advantages.

To transform host cells with an expression vector containing a cDNA encoding prourokinase of the present invention, the competent cell method [J, 5pizizen, Proc, Natl.
Acad, Sci, USA.

44.1072 (1958)], protoplast method [
3, Chang, Mo1. Gen, Genet,
, 168, 111 (1979)], calcium phosphate method [G7ahas, F, L, et, al.
Viroloay, 52, 456 (1973)],
Electroporation [Wong, T., a.
nd Neumann E, BiochetBi
ophys, Res, Commun,, 1 0
7. 584 (1982). Selection of transformants into which the expression vector has been integrated can be performed by conventional methods based on the selection marker contained in the expression vector. Alternatively, for example, a gene encoding a drug resistance marker can be co-transfected with the expression vector, and transformants can be selected based on the drug resistance.

The target prourokinase is expressed by culturing the transformant thus obtained in an appropriate medium under appropriate culture conditions. When the expression vector contains a DNA sequence encoding the dhfr protein, the expression vector copy number is amplified by culturing in a medium containing a dhfr protein inhibitor such as methotrexate or homofolate. The expression level of urokinase increases.

The desired prourokinase derived from human vascular endothelial cells can be obtained by collecting prourokinase from cultured cells or culture supernatants by a conventional method.

<Effects of the Invention> By transforming host cells with an expression vector containing cDNA encoding prourokinase derived from human vascular endothelial cells and culturing the transformant, it is possible to mass-produce a new type of prourokinase. Become. In addition, by using a human vascular endothelial cell-derived cell line as a host cell, prourokinase can be efficiently produced, and this cell line can be used as a host cell for expressing recombinant DNA to express other useful proteins. It can be widely used.

<Example> The present invention will be described in more detail below using reference examples and examples.

Example 1 Preparation of established cell line derived from human vascular endothelial cells (1) Preparation of cell line Enzymatic detachment method from human ligament vein [Jaffee, EA
,, J, C11nical Investigat.
ion, Vol. 52, 2745 (1973)]
Primary culture cells were prepared.

In addition, the following items were considered for cells [1 Eizo, Experimental Medicine,
4 (7), 579 (1986) identified them as vascular endothelial cells.

a, Form of cobblestone structure of knee layer, Presence or absence of Weibel Pa1ade-body: YesC, Presence or absence of contact 1 inhibition: Yesd, Presence or absence of angiotensin converting enzyme: Yese, Lectin binding property: Yes, Presence of keratin: No 2) Subculture cells 1 x 10 ~ 20 x 10' Ce11S/ad
! (The cells were implanted in large numbers, and cultured for 3 days. When the number of cells increased approximately 4 times in 3 days, a trypsin-EDTA mixture was added to collect a monolayer of young cells. A cell-derived cell line was obtained.The culture medium for this cell line was, for example, an oral tube supplemented with 10% heat-inactivated beef tallow serum.
Use ecco's modified MEM or the like. Cultivation is preferably carried out under conditions of 37°C containing 5% CO2.

Example 2 The concentration was decreased from 10% to 5% and culture was continued. Although the proliferation ability was extremely reduced, the culture was continued for about 20 days until the proliferation ability was recovered, and when it became confluent, it was subcultured. Next, the serum concentration was decreased from 5% to 1% and culture was continued.

The proliferation ability was extremely reduced, but it took about 2 hours until the proliferation ability recovered.
Culture was continued for 0 days and passaged twice. Furthermore, the serum concentration was increased to 1%.
Instead, a serum replacement medium, Urt O Sur G (manufactured by IBF Biotechnics), was added at a rate of 3 ay/d, and the culture was continued. Although the proliferation ability decreased, culture was continued for about 20 days until the proliferation ability recovered.
After passage several times, a stable cell line (ECsf15) was finally obtained.

Reference Example 1 (1) Production of Pro-UK from human vascular endothelial cells The human vascular endothelial cells obtained in Example 1 were
Using the cell line obtained in Example 1 with a number of x 10' cells/ae (7) and a diameter of 10, the serum concentration was spread into a plastic petri dish (manufactured by Falcon), and 10% heat-inactivated fetal bovine serum was added. Dulbecco's mod including
Add IFed MEM medium and continue culturing for 3 to 5 days [
Puta. Once the cells were confluent, the medium was diluted with Dulbecco's solution containing 0.03% bovine serum albumin.
's g+odified MEN serum-free medium. After culturing for 4 to 5 days, all the supernatant is collected using a 10-ml plastic bivet (manufactured by Falcon). After that, 10 times of the same serum-free medium as above is added and cultured in the same manner.

If 50 culture dishes are used and this operation is repeated 8 times, approximately 4 liters of supernatant will be obtained.

Note that all of the above operations were performed aseptically. In addition, all culturing was carried out in a carbon dioxide ink 1 betater (manufactured by Tezawasha Co., Ltd.) set at 37° C. and 5% Co 2 .

(2) Concentration of Pro-UK with S-Seph70-Scolumn About 30M1 of 1N hydrochloric acid was added to 4.0OOd of human vascular endothelial cell culture supernatant to adjust the pH to 5.5.

Then, the flow rate lad! /Win and washed the column with 300ml each of 0.16M phosphate buffer (pH 5.5 and pH 6.5) containing 10 KIU aprotinin, then the same! The adsorbed prourokinase A was eluted with S buffer (pH 7.5). Collect 15 d fractions and perform fibrin plating (Stal) hV.
IOcOcci and Staphylococcal
Infections, Ed, by JJelj
aSZewiCZ, Ba5el, S, arger
, 1973, p406-412), and the active ingredient containing the present Pro-UK was eluted in the 20th to 25th samples (see Figure 2).

(Pro-
UK fraction 0.5M Sodium Chloride and 0.01% Tween 80
Zn-chelate toyobal (ID50mX10
Pour the above eluent into a tube (manufactured by Tosoh Corporation) at a flow rate of 1 d/I.
After adsorption with the same buffer, 0.05M containing 0.5M sodium chloride and 0.01% Tween 80
A 30 minute l)H gradient elution was carried out with Tris acetate buffer (pH 7.5-5.5) (see Figure 3).

The active ingredient was 1) eluted as a broad peak from H6,5 to 5.5.

f4) Book p by Zn-chelate rechromatography
ro-UK purified 0.5M sodium chloride and 0.01% ween80
Zn-chelate-5PW (ID7.5am+X7
5am, Tosoh News) including about 1■ of this Pro-UK.
d above fractionated sample at a flow rate of 0.255dj! /lln
After washing with the same buffer, it was eluted with a 5IH Tris acetate buffer containing 0.5 M sodium chloride and o, oi% Tween 80 at a flow rate of 1 ae/min. Elution was performed using a 30 minute pH gradient from pH 7.5 to pH 5.5 (see Figure 4).

This Pro-UK was eluted as a sharp peak at about I)H6. After desalting and freeze-drying, a white powder of about 0.9 cm was obtained.

In this way, the present Pro-UK isolated and purified to a high degree of purity was obtained.

(5) SDS-polyacrylamide gel electrophoresis of this Pro-LIK Pro-UK at 100°C for 5 minutes, 1% SDS, 1%
After reduction treatment with 2-mercaptoethanol, proteins with known molecular weights (urokinase derived from human urine, (L
5DS-polyacrylamide gel electrophoresis together with human urine-derived prourokinase (pro-LIK, manufactured by American Diactustech). Also this Pro-L
In order to check the degree of purification of IK, the S-Sephas fraction obtained in (2) above was also subjected to electrophoresis at the same time (see Figure 5).
. Note that similar results were obtained when the sample was subjected to 5DS-polyacrylamide gel electrophoresis without pretreatment.

From the above results, the molecular weight of this Pro-LIK is approximately 50,000 Daltons, and it has a single-stranded structure, and the Zn-LIK of (4) above
It was confirmed that this Pro-UK was highly purified by chelate rechromatography.

(6) Expression of urokinase activity by plasmin treatment
Dilute with PBS containing 1% rween 80 and add 40μ
m was collected. Add 5 μ! of 1801c (1/d plasmin) and incubate at 37°C.
After 20, 40, and 60 minutes, the reaction was stopped with aprotinin, and the urokinase activity of the reaction solution was determined by reacting the synthetic substrate S-2444, which is a substrate for urokinase, with the above treatment solution and measuring the resulting color using a colorimetric method. (See Figure 6).

A similar experiment was conducted using a known prourokinase derived from human urine (see Figure 7). Based on the above results, this Pr
o-LIK, like known prourokinase, is activated over time by plasmin treatment, and this Pro
-UK was confirmed to be a urokinase precursor (pro-urokinase).

Example 3 cDNA base sequence encoding the present Pro-tJK (1)
Extraction of mRNA from human vascular endothelial cells Reference example 1 (
Sow the subcultured cells in 50 plastic petri dishes (manufactured by Falcon) with a diameter of 10 cII using the method shown in 1).
When the cells became confluent, the medium was changed to serum-free, and on day 19, cells were collected using Cellus Flavor. Total RNA was extracted from these cells by guanidine thiocyanate/CsC1 ultracentrifugation.

(2) Northern hybridization using oligonucleotide probes Known base sequence of prourokinase CDNA derived from human kidney cells [8, Nagai et al., Gene 3
6.183-188 (1985)] and six types of oligonucleotides (see Figure 8).
We used the Northern hybridization method to determine whether it would hybridize with synthesized RNA extracted from cells.

The oligonucleotides shown in FIG. 8 were synthesized, the 5' ends were labeled with 32P, and these were used as probes.

After the above RNA was fractionated by 1.4% agarose gel electrophoresis, it was transferred to a nitrocellulose filter and hybridized, and it was confirmed that it hybridized with these probes.

Purification of f31 mRNA Total RNA was adsorbed onto an oligo dT cellulose column, the column was washed, the mRNA was eluted, and the mRNA that hybridized with the oligonucleotide was collected.

160 μ9 mRNA was obtained from 3.59 total RNA.

(4) Synthesis of cDNA Using a cDNA synthesis kit (manufactured by Amajam), cDNA was synthesized from purified mRNA 15 μ9 using oligo dT as a primer.

f5) Binding of EC0R1 linker to cDNA A cDNA cloning kit (manufactured by Amajam) was used. First, the ECoR1 site inside the cDNA was methylated, EcoR1 linkers were attached to both ends of the cDNA, and then the cDNA was cleaved with ECORI and the cDNA was purified by gel filtration.

(6) Creation of CDNA library of this Pro-LIK The cDNA was combined with λGt11EcoR1 arm, and In
Phage particles were formed by VltrO packaging and a CDNA library was created.

(7) Isolation of a recombinant containing the cDNA of this Pro-UK from a library - The λat11 recombinant containing the cDNA of this Pro-UK was adsorbed to E. coli strain 12 LE392 and plated to remove plaques. formed. After transferring the plaque to a nitrocellulose filter, the filter is alkaline denatured, neutralized, and baked to remove recombinant cD.
NA was immobilized on the filter.

The 5' end of the oligonucleotide shown in Figure 8 was
- Labeled using ATP and hybridized with the DNA immobilized on the above filter. Hybridization and subsequent washing were performed at a temperature 5° C. lower than the Td shown in FIG.

Td was calculated assuming that A and T contribute 2°C and G and C contribute 4°C.

Plaques hybridizing with these oligonucleotides were purified. As a result, seven clones were obtained, one of which was named λtJK-L.

Subcloning λUK using +81M13 vector
-1 was prepared in large quantities, the cDNA was extracted, and a restriction enzyme breakpoint map of the cDNA portion was created (Figure 9).

Cut the cDNA with two types of restriction enzymes to create M13-O18.
Alternatively, subcloning was performed using M13mp19 as a vector.

(9) Base sequence analysis
r method [Sanger, r, etat, Pr
oc, Natl, ^cad, Sci, U, S, ^
, 74.5463-5467 (1977)], the base sequence of the CDNA was determined along the direction of the arrow shown in FIG. The base sequence of the cDNA encoding Pro-UK and the amino acid sequence determined therefrom are shown in FIG.

Example 4 (1) Preparation of expression vector An expression vector was constructed according to the construction scheme shown in FIG.

(Amplification of il P r O-U K CDN A) Using λUKL DNA, which is a recombinant of λot11 having Pro-LIK cDNA obtained in Example 3 (7), as a template, and using otl 1 brimer and reverse brimer. PCR (Polyg+erase Chai
n Reaction) [5aiki
etal in R, 5science, 230. 135
0 (1985)] method.

This was disassembled by AccI[, Stu, and shown in Figure 10 (
The 1.45 kb band shown in b) was fractionated.

In O of Fig. 10, the grid-like part is P″ro-UK
The structural gene encoding is shown, and the shaded part shows the 3' untranslated part.

! Synthesis of DNA for ligating to Pro-LIK cDNA
t) E) Manufactured by Iied Biosystem, 380B
and purified using an OPC column (manufactured by AB 1).

5°G^TCAAGCTT GCCACCATG A
GG GCT CTG CTG GCC3'...
(c) 3go TCGAA CGGTGG TACTCC
CGA GACGACCGG 5'...In the above DNA, the ozak sequence [H, Kozak,
Nature, 308. 241 (1984
)] (dotted line) was inserted. Pr0-UKcDNA
@ i ndll so that the translation region can be extracted later
[The part (underlined part) was introduced.

Specifically, -5' of heavy chain DNAe (30mer)
The terminal is phosphorylated, -heavy chain DNA (c) (34mer
) was added, heated at 100° C. for 5 minutes, and then left at room temperature to allow DNA (c) and DNAO19 to anneal to synthesize double-stranded DNA ((Gl in FIG. 10).

(i) Plasmid I) Construction of SVLUK (i) above
DNA obtained in (t)l was added with D obtained in (iil) above.
N A (0) was added and ligation was performed with T4 DNA ligase. After inactivation of the glue gauze at 68°C for 10 minutes, the 5' end was phosphorylated and Sepharose CL-
Purify the DNA using a 6B spin column and obtain 1.45kb.
DNA (b) on the 5' and 3' sides of the DNA (b)
DNA in which 0 was linked <0 in Figure 10) was obtained.

Plasmid pSVL [manufactured by Pharmacia; Tei+p
leton, D. et al., Hot, Ce.
11. Biol, 4°817 (1984)] was cut with BamH, treated with T4 polymerase, and CI
The 5' end was dephosphorylated by P treatment to obtain an open circular double-stranded DNA (0 in Figure 10). This plasmid ps
The VL has f3 amHI and 5alI sites in the appropriate positions, and the -8 V40 late promoter,
Contains the SV40 late polyazonylesion signal,
It is constructed from SV40 and pBR322.

Open circular double-stranded DNA (F>, the above DNA (E
), ligation was performed using T4 DNA ligase, and the resulting ligation was used to transform Escherichia coli HBIO1 to obtain plasmid psVLUK. After confirming the direction of integration, the transformed E. coli was mass-cultured and subjected to the alkaline method [Birnbois, H, Cand J,
Doly, Nucleic Ac1ds Res.
E, 1513 (1979)].

(M Plasmid 1) Construction of SVLUKdhfr dh
Plasmid DDHFR containing fr (JP-A-1-2851
90) was digested with BamHI, the ends were made blunt by lenow fragment treatment, and a DNA fragment containing dhfr was fractionated by agarose gel electrophoresis.

DSVL was digested with 5alI, and the ends were blunted by treatment with 1einOW7 lacment. 5 of the DNA obtained
After dephosphorylating the ' end and ligating with a DNA fragment containing dhfr, E. coli strain H8101 was transformed. DNA was prepared from the obtained ampicillin-resistant strain, its structure was confirmed, and DNA encoding Pro-LIK and dt
The desired expression vector pSVLUKdhfr containing DNA encoding the lfr protein was obtained.

+2) Transformation of CHO-K 1 cells (ilDNA
Preparation of solution plasmid pNE containing neomycin resistance marker
o [Jorensen, R.A., et al.
,, HolecGen, Genet,, 177,
65 (1979)] 50ona l:coR1F
It was cut and heat treated at 65°C for 10 minutes. There, psVLLJKd hf r-DNA prepared in (1) above was added.
30u9 was added, sodium acetate buffer (pH 5,2) was added to make the concentration 0.3M, 2 times the amount of ethanol was added, and the mixture was left at -80℃ for 20 minutes.
pm, and centrifuged for 10 minutes, and the precipitate was suspended in 70% ethanol. After centrifugation again, ethanol was completely removed and the suspension was aseptically suspended in 450 μl of sterile water. Note that all subsequent operations were performed aseptically.

1ii) Formation of DNA-Calcium Phosphate Precipitate 50d Autoclaved in sterile conical tube 50
0μJHNP (50mHHepes buffer (1) 87
．． 1)/280+nH Sodium chloride/1.51N
(disodium phosphate) and 50μ of the DNA prepared in (i) and filter-sterilized 2.5M calcium chloride.
1 Add the mixed solution dropwise while bubbling, and continue adding 1 after dropping.
Continue bubbling for 30 minutes, then leave for 30 minutes to remove DNA.
- A calcium phosphate precipitate was obtained.

Co-transfect CHO-Kl cells into 1 cell (Il CHO-1 x 10” and 2 x 105
The cells were cultured in F12 medium containing 10% fetal bovine serum in a diameter 6α plastic jar (manufactured by Falcon). In addition, the medium was replaced with fresh medium 5 hours before transfection.

Precipitate DNA-calcium phosphate 1 per petri dish
d to the cultured cells, and after 16 hours, the medium was replaced with fresh one. After 24 hours, the cells were diluted 3 times, and after another 24 hours, the culture medium was diluted with 400μ9/d G4.
The selective medium was replaced with a selective medium containing 18.

After culturing for 2 days, each petri dish was diluted 6 times and subcultured in the above selective medium. On the fourth day of culture, the cells were diluted 3 times and subcultured, and the culture medium was exchanged every 3 to 4 days, and culture was continued for about 40 days.

(Selection of M transformants After co-transfection, morphological changes were observed in the cells from the 3rd molar onwards, a considerable number of cells died on the 7th day, and colonies were observed around the 10th day.

About 15 days later, cloning was performed using the cylinder method. The colonies that appeared were marked under a microscope, and each colony was surrounded by a stainless steel cylinder for cloning.
After washing with PBS (-), 100 μl of 0.25% trypsin solution containing 0.02% EDTA was added. Approximately 5
After incubation at 37° C. for minutes, trypsin was neutralized with the same amount of the above selection medium, the cells were detached by pipetting and seeded on a 96-well plate, and 100 μm of the above selection medium was added.

When each well reached confluency 1 or more, it was passaged into a 48-well plate.
Passage was continued to 4 wells and then to 12 wells.

Once the 12-well plate is confluent,
Each well was subcultured into two 12-well plates, and when it became confluent, one well was subcultured into a 6α-diameter plate. The medium of the other one was changed to F-12 medium containing 0.03% bovine serum albumin, and after 4 days, Pro-UK activity in the culture supernatant was measured by fibrin plate method [5taphylococci and 5taph].
ylococcal summer infections, Ed.
by J, Jeljaszewicz, Ba
5elS, Niarger, p. 406-412 (1
973)].

Particularly high activity was observed in 3 clones among 48 clones (see Table 1).

Table 1 Clone number Pro-UK activity (IU/d) CHO-
LJK8 32.0 CHO-tJKlo 39.5CHO-UK1
9 49.5 f3) Transformation of E Cs f 15 cells (i)
Cell Culture Unless otherwise specified, the cell culture medium used in this procedure was a sterilized Dulbecco's modified MEM (hereinafter abbreviated as DMEM) medium containing 10% fetal bovine serum, and all culture operations were performed aseptically. went. The day before transfection, EC5f15 cells were directly transfected with 2X10 per W6tx>v-le.
．． The cells were seeded at 5x105 cells, and the medium was replaced with fresh medium 3 times 5 hours before transfection.

1iii Preparation of DNA solution psVLLIKd h f r- constructed in (1) above
Add 1/10 volume of 3M sodium acetate buffer (pH 5.5) and 2.5 times volume of 20°C ethanol to a solution containing 3 μ9 of DNA, leave at -70°C for 15 minutes, and add 1500
Centrifugation was performed at 0 rpm for 10 minutes.

The obtained precipitate was suspended in 95% ethanol and centrifuged again under the same conditions to recover DNA. After vacuum drying, 120
The suspension was aseptically suspended in μl of distilled water and used for transfection. From now on, all operations were performed aseptically, using sterilized instruments and reagents.

(2) Transfection Transfection was performed by the calcium phosphate method, and the reagent used was Cellfect Transfection Kit (manufactured by Pharmacia).

buHe containing 0.5M calcium chloride 0.05M Repes buffer to the DNA solution prepared in (ii) above
120 µO of rA was added and thoroughly mixed using a portex mixer. After standing at room temperature for 10 minutes, 0.28M sodium chloride, 0.05M Heoes buffer, 0.751
Buyer B containing M Sodium Phosphate and 0,'75i+H Disodium Phosphate.

240 μm was added, mixed again for a few seconds using a portex mixer, left at room temperature for 15 minutes, and then dropped onto the cultured cells prepared in (i) above.

4.5 hours after transfection, all the medium was removed by suction, and 1.5 d of PBS (-) (manufactured by Flow Laboratories) containing 15% glycerol was added and left for 1 minute. Subsequently, the cells were washed twice with serum-free DMEM medium, and a cell culture medium was added 5 times, followed by culturing. After culturing for 2 days, each petri dish was diluted 6 times and subcultured. At this point, the medium was replaced with a selective medium containing 20 nt4 methotrexate and culture was performed. On the fourth day of culture, the cells were diluted 3 times and subcultured, and the medium was exchanged every 3 to 4 days.
Culture was continued for about 40 days.

(Selection of M transformants After transfection, morphological changes were observed in the cells from the 9th molar onwards, and on the 16th day, the cells died, and colonies were observed around the 30th day.

About 40 days later, cloning was performed using the ring method.

Mark the colonies that appeared under a microscope, surround each colony with a stainless steel ring for cloning, and
After washing with BS (-), 0 containing 0.02% EDTA
．． 100 μl of 25% trypsin solution was added. After incubating at 37°C for about 5 minutes, trypsin was neutralized with the same amount of the above culture medium, the cells were detached by pipetting, and seeded in a 96-well plate, and then the above selective medium 1 was added.
00 μl was added.

When each well became confluent, it was passaged into a 48-well plate, and each time it became confluent, 24 wells were passaged.
Passage continued to wells and 12 wells.

When the 12-well plate reached confluency, each well was subcultured into two 12-well plates, and when the 12-well plate reached confluency, one well was subcultured into two 12-well plates with a diameter of 6 CI.
It was subcultured to R Schiare. The medium of the other plate was replaced with DMEM medium containing 0.03% bovine serum albumin, and after 4 days, Pro-LIK activity in the culture supernatant was measured by fibrin plate method.

Particularly high activity was observed in 3 clones among 63 clones (see Table 2).

Table 2 Clone numbers of human vascular endothelial cells transformed cells pro-LIK activity (I U/d) EC
5f15-25 83.0 ECsf15-31 79.2 ECsf15-33 82.0 (v) P r O-LI K production increases over time Particularly high Pro-LJK activity in the culture groove EC5f15-
No. 25 was cultured in the above serum-free medium.

4 x 10' ~ 8 x 10' cells/d
(7) The cells were seeded in 12-well plates and cultured for 44 days using DMEM medium containing 0.03% bovine serum albumin and 10 1 KLI/id aprotinin. Replace the medium with fresh medium every 4 days and check for increase in activity (see Table 3).

Table 3: Day 4 of human vascular endothelial transformed cells Day 44 EC5f 83.0 20
7.6 (4) Confirmation of the product of the genetic recombinant (it Adjustment of the culture supernatant) EC5f15-, which has particularly high pro-UK activity in the culture supernatant
No. 25 was cultured in the above serum-free medium.

4X 10 ~ 8X 10' cells/ad! 0.03%
The cells were cultured for 28 days using a DMEM medium containing bovine serum albumin and 10 IKU/d aprotinin.

The conditioned medium was replaced with fresh medium every 4 days and stored at 4°C.

Pro-UK was purified in the same manner as in Reference Example 1 using an S-cephalometric ram and 7n-chelate-5PW.

(ii) 5DS-PAGE of PA Purified Pro-LJK was analyzed by 5DS-PAGEr. This Pro-UK, a protein with a known molecular structure, human urine-derived urokinase (manufactured by American Diagtustica), and human urine-derived Pro-LIK (manufactured by American Diagtustica) were each incubated at 100°C for 5 minutes. 1%SO8゜1%2
- After reduction treatment with mercaptoethanol,
It was subjected to 5DS-PAGE. The electrophoresis is the chemical agent-5O.
A 3-PAGE apparatus and a 4/20% gradient gel manufactured by the same company were used. The results are shown in Figure 11.

From the above results, it was found that this transformed cell produced Pro-UK.
was confirmed to be of the pro-urokinase type.

[Brief explanation of drawings]

Figure 1 shows human vascular endothelial cell-derived prourokinase (
The base sequence of the cDNA encoding Pro-UK) and the amino acid sequence determined therefrom are shown. FIG. 2 shows the elution profile of the supernatant of human vascular endothelial cell culture when it was subjected to S-Sepha O-Slum chromatography. FIG. 3 shows the fractionation profile of Pro-UK by Zn-chelate Toyovar chromatography. Figure 4 shows the purification of Pro-LJK by zn-chelate-5PW chromatography. FIG. 5 shows the results when this Pro-UK was subjected to electrophoresis together with other proteins of known molecular weight. FIG. 6 is a graph showing the urokinase activity of the present Pro-UK after plasmin treatment. FIG. 7 is a graph showing the urokinase activity of human urine-derived prourokinase after plasmin treatment. Figure 8 shows the synthetic oligonucleotide probe used to detect and isolate mRNA encoding Pro-UK. Figure 9 shows the restriction enzyme map of the cDNA encoding Pro-UK and the dideoxy chain ternna.
The direction of the seek engine according to the tion method is shown. FIG. 10 is a diagram showing the construction scheme of the development vector pSVLUKd h f r. FIG. 11 shows the results when the culture supernatant of psVLUKd hf rt'-transformed human vascular endothelial cells was purified and subjected to electrophoresis.

Claims (7)

[Claims] (1) A method for producing prourokinase, which comprises transforming a host cell with an expression vector containing a cDNA encoding prourokinase derived from human vascular endothelial cells, and culturing the resulting transformant. (2) The method for producing pro-urokinase according to claim 1, wherein the cDNA encoding pro-urokinase derived from human vascular endothelial cells has the DNA base sequence shown in FIG. (3) The method for producing prourokinase according to claim 1 or 2, wherein the host cell is an established cell line derived from human vascular endothelial cells. (4) The method for producing prourokinase according to claim 1 or 2, wherein the host cell is an established cell line derived from human vascular endothelial cells that can be cultured in a serum-free medium. (5) Claims 1 to 4, wherein the expression vector contains a DNA sequence encoding dihydrofolate reductase protein.
A method for producing prourokinase according to any one of paragraphs. (6) Recombinant D consisting of established cell line derived from human vascular endothelial cells
Host cells for NA expression. (7) The host cell according to claim 6, wherein the human vascular endothelial cell-derived cell line is a human vascular endothelial cell-derived cell line that can be cultured in a serum-free medium.