JPH05252951A - Production of procollagenase - Google Patents

Abstract

PURPOSE:To safely produce a procollagenase useful as a reagent for research in the field of medical chemistry, etc., at low cost in a high yield by adding a specified procollagenase-production promoter to a culture medium when culturing a cell for collecting procollagenase. CONSTITUTION:A cell [e.g. human-derived Detroit-551 (ATCC-CCL-110)] is cultured in a culture medium containing a salt selected from a sulfate (e.g. sodium sulfate) of 2 to 80mM final concentration, a nitrate (e.g. ammonium nitrate) of 0.5 to 100mM and an ammonium salt (e.g. ammonium acetate) of 0.5 to 20mM. The culture medium is prepared preferably, e.g. by adding amino acids and/or vitamins to a serum- and protein-free basal medium, further adjusting pH and subsequently adding 0.5 to 10vol.% (fetal) bovine serum thereto.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a process for producing procollagenase useful as a reagent for research in medicinal chemistry, biochemistry and pharmacy.

[0002]

2. Description of the Related Art Procollagenase is a precursor of collagen degrading enzyme (collagenase) and has a molecular weight of 52,000 or
57,000 are known [Biochemistry (Biochemistry
Chemistry), 27, 6751, 1988]. They are,
Interstitial collagenase (hereinafter referred to as collagenase unless otherwise noted) that is activated by proteolytic enzymes such as plasmin and trypsin or mercury compounds to decompose interstitial collagen (type I, type II and type III collagen) .)

Collagenase is considered to regulate the metabolism of collagen (interstitial collagen), which is abundant in connective tissues of animals, and causes abnormal accumulation of collagen. Cirrhosis, arteriosclerosis, pulmonary fibrosis and It has been noticed and studied in relation to rheumatoid arthritis, periodontitis, corneal ulcer, etc., in which keloid or collagen degradation is observed [eg American Journal of
Pathology (American Journal of Pathology), Vol. 92, 5
See page 09, 1978].

[0004] Procollagenase, which is a precursor of collagenase, is also attracting attention for the elucidation of the above-mentioned respective pathological conditions [The Journal of Biological
Chemistry (The Journal of Biological Chemistr
y), 258, 9374, 1983]. Therefore, high-purity procollagenase is required for medicinal, biochemical, and pharmaceutical studies of the above-mentioned pathological conditions, and an efficient production method thereof is desired.

It is already known that human fibroblasts or fibrosarcoma cells produce collagenase and its precursor in vitro, and procollagenase has been obtained from the culture medium of these cells [Biochem (Biochem
istry) 16, 1607, 1977/25, 4750, 1986
/ Volume 27, page 6751, 1988 / Proceedings of the National Academy of Sciences
Of the United States of America (P
roceedings of the National Academy of Sciences of
the United States of America), 83, 3756, 1986
See year].

The present inventors have already clarified a method for producing procollagenase by using a serum-free protein-free medium (JP-A-3-103178).

[0007] In producing procollagenase using the above-mentioned known method, it is necessary to improve the procollagenase-producing ability of the cells used in order to increase the productivity.
As substances capable of enhancing the procollagenase-producing ability of cells, cytokines such as interleukin 1, tumor necrosis factor (TNF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) and hormones have been heretofore used. Ball ester and the like are known. However, these cytokines are expensive and the manufacturing cost is high. Also, phorbol ester is a carcinogen and its use is dangerous.

[0008]

Therefore, an object of the present invention is to provide a method for efficiently producing procollagenase by adding an inexpensive and safe procollagenase production promoting substance to a cell culture system. is there.

[0009]

[Means for Solving the Problems] The above-mentioned object is to obtain a procollagenase by culturing cells, and as a medium composition, the final concentration is 2 to 80 mM sulfate, 0.5 to 100 mM nitrate or 0.5 to 20 mM ammonium. This is achieved by a method for producing a procollagenase, which comprises one or more selected from the group consisting of salts.

The method for producing procollagenase of the present invention will be described below.

The sulfate used in the present invention is, for example, sodium sulfate, magnesium sulfate, ammonium sulfate or potassium sulfate, the nitrate is potassium nitrate, sodium nitrate, ammonium nitrate or the like, and the ammonium salt is ammonium acetate, etc. Examples thereof include ammonium tartrate.

It is essential that the concentration of sulfate in the medium is 2 to 80 mM, preferably 2 to 60 mM. It is important that the concentration of nitrate in the medium is 0.5 to 100 mM, preferably 2 to 80 mM. It is essential that the concentration of ammonium salt in the medium is 0.5 to 20 mM, and preferably 1 to 10 mM.

The method of adding the sulfate, nitrate or ammonium salt to the medium is not particularly limited and may be directly mixed with the medium.

The cells used in the present invention include:
Fibrosarcoma cells and fibroblasts, such as human-derived Detroit 55
1 (ATCC CCL 110), Detroit 548 (ATCC CCL 116), BUD
8 (ATCC CRL 1554) etc. can be used.

Examples of the medium include Ham's F-12 and Eagle
Normal serum-free protein-free basal medium such as MEM, RPMI-1640, MEM Dulbecco's liquid medium or the like, or a mixed medium of two or more kinds, preferably, amino acids and / or vitamins are added to these mediums to adjust the pH to 6.5- An animal, such as bovine or fetal calf serum, was added to the one adjusted to 7.4.
1 to 20% by volume, preferably 0.5 to 10% by volume can be used. If necessary, auxiliary additives such as lactalbumin hydrolyzate and sodium pyruvate can be added.

Antibiotics can be added to these media in order to prevent contamination with bacteria and mycoplasma. Further, in order to adjust the pH of the medium, inorganic salts such as sodium carbonate and sodium hydrogen carbonate may be added or carbon dioxide gas may be dissolved.

Streptomycin sulfate, kanamycin sulfate and the like have been conventionally used as antibiotics, but the compounding amount thereof is as small as 2 mM or less, and the effect of the present invention is not exhibited.

Normal fibroblasts usually have high procollagenase productivity when cultured at a cell density of 10 ⁴ cells / cm ^{2 of} flask bottom or more. Therefore, the cells previously grown in the above-mentioned medium are collected, preferably 1 × 10 ⁴ to 1 × 10 ⁵
Cell density of cells / cm ² , more preferably 2 × 10 ^{4 to} 8 × 10 ⁴ cells / c
Cells are seeded at a cell density of m ² and sulfates, nitrates or ammonium salts are added, and usually 4 to 20 days, preferably 6 to
Incubate for 14 days.

Needless to say, it is also possible to start the culture at a cell density below the above-mentioned level to allow the cells to grow, and to further culture for 4 to 20 days, preferably 6 to 14 days after reaching a cell density higher than this. Yes.

Next, cells are removed from the culture broth by an ordinary separation means such as centrifugation, filtration and suction, and procollagenase is separated and purified from the resulting culture supernatant by column chromatography. Separation and purification by column chromatography is usually performed in two steps. Procollagenase eluted from the column is detected by a known procollagenase amount measuring method, and fractionated using this as an index.

In the first-stage column chromatography, an ordinary cation exchanger such as CM-Sepharose CL-6B ^™ (Pharmacia) or a heparin group-specific adsorbent such as heparin Sepharose CL-6B ^{™ is used.}
The culture supernatant is passed through a column filled with (Pharmacia) and eluted with the following eluents to fractionate the fraction of crude procollagenase.

The eluent for column chromatography packed with a cation exchanger contained CaCl ₂ and 0.01 to 0.1% by volume.
Tris-HCl buffer containing non-ionic surfactant
_{2. Using} a Tris-HCl buffer (pH 7.8) containing a nonionic surfactant and NaCl, a linear NaCl concentration gradient is applied to elute the procollagenase. Polyoxyethylene lauryl ether is preferably used as the nonionic surfactant. Procollagenase has a NaCl concentration of 0.3 to 0.
Elute at 5M.

The same buffer as above can be used as an eluent for column chromatography packed with a heparin group-specific adsorbent, but in this case, procollagenase is eluted at a NaCl concentration of 0.4 to 0.6 M. To do.

Next, for the second-stage separation and purification, the crude procollagenase fraction eluted as described above is concentrated with a molecular sieve membrane filter, if necessary, and then purified with metal chelate affinity column chromatography. In this column, for example, metal chelating Sepharose 6B ^™ (
A product obtained by chelating zinc in Pharmacia (hereinafter abbreviated as zinc chelating sepharose) is filled and used.

The eluent contains an acetate buffer (pH about 4.8) containing NaCl, CaCl ₂ and a nonionic surfactant (eg polyoxyethylene lauryl ether), and NaCl, CaCl ₂ and a nonionic surfactant. Using 2- (morpholino) ethanesulfonic acid monohydrate buffer (pH about 6.8),
Elute from the column with a gradient (gradual decrease in pH).

Procollagenase has an eluent pH of about 5.3.
It is eluted when it reaches the vicinity, so this fraction is collected and, if necessary, a molecular sieve membrane such as DIAFLO ^™ YM-5 or YM-1.
A membrane such as 0 (all manufactured by Amicon) is used for concentration, and if necessary, the pH is adjusted to about 7.5 to obtain an aqueous solution of procollagenase. An aqueous solution of procollagenase is usually frozen and stored.

[0027]

According to the method of the present invention, the productivity of procollagenase per cell can be significantly increased.
Further, the sulfate, nitrate or ammonium salt used in the present invention contains procollagenase at a low cost and in a high yield, as compared with growth factors such as TNF and PDGF which are known to enhance procollagenase-producing ability. It has the advantage of being manufacturable.

[0028]

EXAMPLES The present invention will be described in more detail with reference to examples below, but prior to that, various measuring methods and the like will be described.

(Method for measuring the amount of procollagenase)

Definition of Procollagenase Amount Procollagenase is activated by trypsin by the following procedure, and the amount of collagenase generated to decompose 1 μg of type I collagen per minute is defined as 1 unit of procollagenase.

Method for activating procollagenase A sample solution is dissolved in a measurement buffer solution to obtain about 0.1 to 0.7 unit / m 2.
Prepare a solution of 1 and use this as the test solution. Next, test solution 50
Trypsin (Type 12, manufactured by Sigma) 20 μl (concentration 1 m
Adjust with g / ml and measurement buffer. ) Is added and the mixture is added at 35 ° C for 5
After incubating for 30 minutes, 30 μl of soybean trypsin inhibitor (manufactured by Merck & Co., Inc.) (concentration 3 mg / ml, adjusted with measurement buffer) is added to inactivate trypsin to obtain a collagenase solution. The measurement buffer is 0.2M NaCl,
5 mM CaCl ₂ , 0.05% by volume Brij-35 (polyoxyethylene
23 Lauryl ether product name, abbreviated as Brij below) and
50 mM Tris-HCl buffer containing 0.02% by volume NaN ₃ (pH 7.
Means 5).

Method for measuring collagenase activity Substrate solution obtained by labeling bovine type I collagen with fluorescein isothiocyanate, that is, FITC-collagen (manufactured by Cosmo Bio Inc.) in 0.01N acetic acid (concentration 1 mg / ml)
And the activity (unit / ml) of the collagenase solution is measured according to the method of Nagai et al. (Inflammation, Vol. 4, p. 123, 1984).

In the following examples, cells were cultured in a medium containing a sulfate, a nitrate or an ammonium salt, and the procollagenase production promoting effect was confirmed. The production amount of procollagenase shown in the examples is a value obtained by adding the standard error to the average value measured three times.

Example 1 (Sulfate) Minimum Essential Medium (Dainippon Pharmaceutical Co., Ltd., 10-101) 1
The final concentration of 0.6 g was 0.1% by weight lactalbumin enzyme hydrolyzate (L-9010, Sigma), 1% by volume Non Es
sential Amino Acid (Dainippon Pharmaceutical Co., 16-810), 1 mM
Sodium pyruvate (Dainippon Pharmaceutical Co., 16-820), 1.
2% by weight sodium hydrogen carbonate and 50 mg / l streptomycin sulfate were added, and distilled water was added to make 1 l, and then carbon dioxide gas was blown into the mixture to adjust the pH to about 7 (hereinafter, abbreviated as MEM medium).

Fetal calf serum (Irvine Scient
ific) was added so as to be 10% by volume (hereinafter abbreviated as MEM-10FBS medium) or 0.6% by volume (hereinafter abbreviated as MEM-0.6FBS medium). This MEM medium contains about 0.8 mM sulfate ion.

Normal human fibroblast cell line derived from Caucasian female skin
[Detroit-551 (ATCC CCL 110)] in MEM-10FBS medium
Prepare 1 × 10 ⁵ cells / ml, and use 3 6-well plates each.
2 ml was seeded and cultured at 37 ° C. for 24 hours under 5% carbon dioxide gas and saturated steam.

Pore size of 1M sodium sulfate aqueous solution
It was sterilized by filtration with a 0.2 μm nitrocellulose membrane (manufactured by Advantech Toyo Co., Ltd., DISMIC-25) and diluted with MEM-0.6FBS medium to prepare 0, 10, 20, 40, 60, 80 mM.

24 hours after seeding the cells, the culture solution was removed and M
After washing twice with EM-0.6FBS medium, the medium was replaced with 2 ml of the same medium containing sodium sulfate adjusted to each concentration, and cultured for 6 days in the same manner to obtain a culture supernatant.

0.25 ml of the obtained culture supernatant contained 10 mM Tris-hydrochloric acid buffer solution (at 4 ° C.) containing 1 mM CaCl ₂ and 0.05% by volume Brij.
The pH was adjusted to 7.8, which will be abbreviated as CM-A buffer hereinafter. ) 1.
CM-Sepharose C added with 75 ml and equilibrated with the same buffer
The column was filled with 0.5 ml of L-6B ^™ (Pharmacia).

After removing the inhibitor by washing with 2 ml of CM-A buffer containing 0.125 M NaCl, 0.5 M
The procollagenase was collected by elution with 2 ml of CM-A buffer containing NaCl. Table 1 shows the results of measuring the amount of procollagenase contained in this procollagenase solution by the method described above.

As can be seen from Table 1, the production of procollagenase was promoted by the addition of sodium sulfate.

[0042]

[Table 1]

Example 2 (Sulfate) As in Example 1, potassium sulfate and magnesium sulfate were added to the normal human fibroblast cell line culture system, and the amount of procollagenase in the obtained culture supernatant was quantified. The control (no addition) was 4.5 ± 0.0 units / ml, whereas 20 mM
7.0 ± 0.6 unit / ml when potassium sulfate is added,
When 20 mM magnesium sulfate was added, the amount was 12.2 ± 2.2 units / ml, and it was found that the production of procollagenase was promoted.

Example 3 (Ammonium Sulfate) As in Example 1, ammonium sulfate was added to a normal human fibroblast cell line culture system, and the amount of procollagenase in the resulting culture supernatant was quantified. The results are shown in Table 2. It was It was 22.5 ± 2.8 units / ml in the control (no addition), whereas it was 43.9 ± 5.5 units / ml in the case of adding 10 mM ammonium sulfate, showing that the production of procollagenase was promoted.

[0045]

[Table 2]

Example 4 (Ammonium salt) As in Example 1, ammonium acetate, ammonium tartrate and ammonium nitrate were added to a normal human fibroblast cell line culture system to quantify the amount of procollagenase in the obtained culture supernatant. The results obtained are shown in Table 3. 2 for control (no addition)
While 1.6 ± 5.0 units / ml, 35.9 ± 2.9 units / ml with 2 mM ammonium acetate, 48.1 ± 14.7 units / ml with 1 mM ammonium tartrate, 36.9 ± 4.1 units / ml with 2 mM ammonium nitrate. It was shown that the production of procollagenase was promoted.

[0047]

[Table 3]

Example 5 (Nitrate) As in Example 1, sodium nitrate was added to a normal human fibroblast cell culture system, and the amount of procollagenase in the obtained culture supernatant was quantified. The results are shown in Table 4. It was Control (no addition)
It was 8.5 ± 1.3 units / ml, whereas the addition of 80 mM sodium nitrate showed 17.9 ± 5.2 units / ml, indicating that the production of procollagenase was promoted.

[0049]

[Table 4]

Example 6 (Nitrate) In the same manner as in Example 1, potassium nitrate was added to a normal human fibroblast cell culture system, and the amount of procollagenase in the obtained culture supernatant was quantified. The control (no addition) had 15.8 ± 3.5 units / ml, whereas the sample containing 20 mM potassium nitrate showed 19.5 ± 2.7 units / ml, which indicates that the production of procollagenase is promoted.

Example 7 (Sulfate) Human normal fibroblasts Detroit-551 (ATCC CCL 110) were added to ME.
Prepare 10 ⁵ cells / ml with M-10FBS medium, and add 16 ml of this preparation to ² flasks (bottom area 75 cm ² ).
The cells were cultivated at 37 ° C for 1 day in an atmosphere of% air-5% carbon dioxide gas.

After static culture for 1 day, the culture supernatant was aspirated and discarded, and MEM-0.6 containing sodium sulfate at a final concentration of 60 mM.
Add FBS medium to two flasks with 16 ml each, and add 95% air-5
Incubation was carried out at 37 ° C. for 6 days in an atmosphere of% carbon dioxide gas.

The culture solution was collected and centrifuged (1000 rpm, 5 minutes) to obtain about 31 ml of the culture supernatant. CM-A buffer solution was added to make 100 ml, and then equilibrated with the CM-A buffer solution. C
It was applied to a column (2.46 cm x 18 cm, packing volume 85 ml) packed with M-Sepharose CL-6B ^™ (Pharmacia).

After washing with CM-A buffer, CM-A buffer
Using 250 ml and 250 ml of CM-A buffer containing 0.7 M NaCl, a linear concentration gradient method was performed at a flow rate of 40 ml per hour. Procollagenase was eluted when the NaCl concentration was 0.3-0.5M. When the activity at this time was measured by the method described above, it was 1100 units (concentration: 9.5 units / ml).

Next, this procollagenase solution was added to DIAFLO ^™.
Concentrate to 7.1 ml with YM-10 (Amicon) and MES
It was applied to a column (1.2 cm x 16 cm, packing volume 18 ml) packed with zinc chelating sepharose equilibrated with -A buffer.

After thoroughly washing the column with MES-A buffer,
Elution was performed with 45 ml of acetate buffer and 45 ml of MES-A buffer at a flow rate of 13 ml per hour with a pH gradient (decreasing pH). Procollagenase was eluted as a single peak at pH around 5.3. This fraction was collected to obtain 6.1 ml of a procollagenase solution (concentration: 160 units / ml).

Example 8 (Nitrate) 5.8 ml of a procollagenase solution (concentration 150 units / ml) was obtained in the same manner as in Example 7 except that cells were cultured in a medium containing ammonium nitrate at a final concentration of 2 mM.

Test Example (1) Estimation of Molecular Weight The molecular weight was measured by slab electrophoresis (6% gel) of SDS-polyacrylamide gel according to the method of Lemley (see Nature, Volume 237, page 680, 1970). As a standard molecular weight marker, rabbit muscle phosphorylase b (molecular weight 94000), bovine serum albumin (molecular weight 68000), egg white ovalbumin (molecular weight 43000), and bovine erythrocyte carbonic anhydrase (molecular weight 30000) were used.

(2) Examination of activation by para-aminophenyl mercuric acetic acid (APMA) The above procollagenase solution was replaced with a measurement buffer, and after concentration, a solution of about 200 units / ml was prepared, and 140 μl of this was added to para-aminophenyl Add 20 μl of 8 mM mercury acetic acid solution (prepared by dissolving in measurement buffer) and incubate at 35 ° C for 2 hours. Then, the collagenase activity is measured by the method described above.

(3) Measurement of the substrate specificity of collagenase activity and the cleavage pattern of collagen obtained by activation The collagenase solution obtained by activation by incubation with APMA by the method of (2) above was treated with a measurement buffer. 60 units / m
Dilute to 10 μl and add 10 μl to each of type I, type II and III
Type soluble collagen measurement buffer solution (concentration 1.5 mg / m
l) Add to 100 μl, incubate at 37 ℃ for 4 hours, and electrophorese this solution as in (1) above.

(4) Examination of inhibitors For measurement, a collagenase solution obtained by activating with trypsin as described in (1) above or a collagenase solution obtained by activating with APMA as described in (2) above was used for measurement. 0 with buffer.
Dilute to 5 units / ml, and add 50 μl of this to the inhibitor solution [ethylenediamine 4 disodium acetate solution (dissolved in measurement buffer, concentration 20 units / ml), orthophenanthroline solution (dissolved in measurement buffer, concentration 20 units / ml) or a solution of tissue metalloproteinase inhibitor (dissolved in measurement buffer,
Concentration 40 units / ml)] 50 μl is added, and the collagenase activity is measured by the method described above to determine the inhibitory ability.

In addition, a tissue metalloproteinase inhibitor [The Journal of Biochemistry (The Jou
rnal of Biochemistry), 254, 1938, 1979], human fibroblast WS-1 (ATCC CRL 1502) was cultured, and CM sepharose CL-6B ^™ , zinc chelating sepharose and G-3000 SW-. XL (made by Toyo Soda)
Was prepared by column chromatography using.

The purified collagenases obtained in Examples 7 and 8 were identified as procollagenase, which is a precursor of collagenase, from the physicochemical and biochemical properties shown below.

Molecular weight: 52000. However, there are only 57,000 molecular species, which are presumed to be glycosylated [Proceedings of the National Academy].
Proceedings of the National Acade
my of Sciences of the United States of America),
83, 3756, 1986].

Activation: It does not show collagenase activity as it is, but is activated by trypsin and para-aminophenylmercuric acetic acid to produce collagenase having a molecular weight of 43000. However, a small amount of collagenase with a molecular weight of 47,000 presumed to be a sugar chain-bound type was produced.

Substrate specificity of collagenase activity generated by activation: Soluble collagens of type I, type II and type III were hydrolyzed and cleaved to a chain length of 3: 1.

Inhibitors: Collagenase produced upon activation was ethylenediaminetetraacetic acid disodium, orthophenanthroline and human fibroblast WS-1 (ATCC CRL 150).
2) Inhibited by a tissue metalloproteinase inhibitor.

Claims (1)

[Claims] 1. When culturing cells to obtain procollagenase, the medium composition is selected from the group consisting of a sulfate having a final concentration of 2 to 80 mM, a nitrate having a concentration of 0.5 to 100 mM, and an ammonium salt having a concentration of 0.5 to 20 mM. A method for producing a procollagenase, which comprises one species or two or more species.