JPS62259585A - Separation of single stranded t-pa and double stranded t-pa - Google Patents

Abstract

PURPOSE:To separate a single-stranded t-PA and double stranded t-PA, by bringing an aqueous medium containing the single-stranded t-PA and double- stranded t-PA once into contact with a carrier carrying ETI to adsorb the t-PA and changing the pH of the eluate. CONSTITUTION:A cell culture fluid is passed through a column immobilizing STI which is a Kunitz type inhibitor forming soybean seeds different from ETI in that t-PA (tissue plasminogen activator) is not inhibited to remove a proteolytic enzyme of the type bonding to the ETI. The unadsorbed solution is then passed through the ETI column to adsorb the t-PA and impure proteins are washed and removed to elute the above-mentioned t-PA within a pH region where the single stranded t-PA is eluted. Then single stranded t-PA and double- stranded t-PA or double stranded t-PA is eluted within a pH region where the single-stranded t-PA and double stranded t-PA are eluted to carry out separation and purification.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides for the production of single-chain t-PA and double-chain t-PA from a mixture containing terminal-chain t-PA (tissue plus nogen activator) and double-chain t-PA. It relates to a method of separation and purification.

In the present invention, [! TI, that is, [Erythrina latissima (leguminous plant)
The immobilized Kunisotsu-type inhibitor is produced in the seeds of Erythrina ma, broad-leaved Erythrina) and other Erythrina species and is an inhibitor of trypsin, plasmin, and t-PA, but does not act on urokinase.

Although it is known that this inhibitor can be applied to the purification of t-PA (Japanese Patent Laid-Open No. 118717/1989), it is not known what will happen if two different buffers are used for this inhibitor as in the present invention. Not yet.

As a result of various studies on substances with t-PA activity, we learned that it consists of a terminal chain and a double chain, and created the method of the present invention based on the need to separate and purify these separately. reached.

As a known method to obtain only one 6Jt-PA,
There is a method in which a protease inhibitor is added during cell culture, and a step of separating and purifying the cells from the culture solution is performed in the presence of the protease inhibitor. However, this method cannot completely prevent the transition from single-stranded t-PA to double-stranded t-PA, and is therefore insufficient as a means.

A method for separating single-chain t-PA and double-chain t-PA in the process of separating and purifying t-PA is a method using an immobilized monoclonal antibody that specifically binds to the -terminal chain t-p^. is also known. However, the purification method using immobilized monoclonal antibodies cannot be said to be a method that is sufficiently reproducible in terms of adsorption capacity for t-PA, stability during use, and practicality, and the molecular weight that reacts with anti-t-PA antibodies. It has the disadvantage that it cannot separate proteins of 110,000±20,000 Daltons.

According to the method of the present invention, separation of single and double strands of t-PA is achieved, but depending on the test sample, a small amount of trypsin-like enzyme may be present, which may lead to ET.
By adsorbing and desorbing T, it can become a disturbing factor for the separation of the present invention. For this purpose, it is advisable to remove these impurities in advance as a pretreatment.

As a method for this removal, a Kunisot-type inhibitor produced in soybean seeds, which has approximately the same molecular weight as STI, ie, ETT, and has 80% or more homology in amino acid sequence, may be used (immobilized).

Although STI is similar to ETT in terms of specificity, STI
does not inhibit t-PA. Therefore, when both are used in combination, it is possible to provide the same selectivity as the immobilized anti-human t-p^ monoclonal antibody. i.e.) t-
A cell culture medium containing PA is passed through an immobilized STI column, and among the trace amounts of impure fluorolytic enzyme contained in the culture medium, E
The type of protease that binds to TI is bound and removed, and the non-adsorbed solution is passed through an ETI column to adsorb t-PA.
After impure fluorescent matter is then washed away, single-stranded and double-stranded t-PA are separately eluted by changing the pH of the elution solvent.

The present invention is applicable regardless of the type of cell containing t-PA. In other words, single-stranded and double-stranded IJjt-P can be obtained from melanoma cells, normal human cells, and cells into which the human l-t-PA gene has been incorporated using genetic recombination techniques.
Separation and purification of A is possible. Moreover, regardless of the composition of the culture medium, it is possible to separate and purify single-stranded and double-stranded t-P^ from a serum-added medium as well as from a serum-free medium.

This will be explained below using human t-p^ as an example.

〔Example〕

Example 1 Preparation of affinity reagent (ETI trial cutting): The ETI reagent used in the present invention was prepared and used in a Sepharose column as follows.

Erythrina latissima seeds are grown as Jou-be.
It was collected and processed according to the method of rt) et al. Seeds were ground, defatted and extracted with 0.5 mol/JNaCl aqueous solution overnight at 10°C.

The extract was centrifuged and the target product was recovered from the supernatant by ammonium sulfate precipitation, followed by Sephadex G50,
Chromatographed on DEA-cellulose and DEA-Sepharose.

The final purified product is 0.1χ sodium dodecyl sulfate (SO
5) When subjected to electrophoresis on a 15% polyacrylamide gel containing 1i22. It migrated as a single band of ooo Dalton.

The purified product (26 mg) was bound to 5 ml of commercially available cyanogen bromide activated agarose in a conventional manner.

The affinity reagent is NaCl 0.4 mol/Il, 0.1
Equilibrated against PH 7.4 phosphate buffer containing X Triton X-100 and 0.02% sodium azide stabilizer.

This affinity reagent was packed into a 5 ml column made from the cylinder of a disposable plastic syringe.

Preparation of affinity reagent (STI reagent): Preparation of the STI reagent used in pretreatment is as follows.

Preparation of 5TI-Sepharose from the above-mentioned ET from soybean seeds
Kunitz-type inhibitor (STI) 25 purified by grinding, extraction, ammonium sulfate precipitation, and chromatographic purification as in the case of I.
mg was obtained and bound to 5 ml of commercially available cyanogen bromide-activated agarose in a conventional manner.

The affinity reagent is NaC1O, 4 mol/10.1χTrit
Equilibration was performed against phosphate buffer containing onX-100 at pl+7.4. This affinity reagent was packed into a 5mlO column made from the cylinder of a disposable plastic syringe.

Using these, -heavy chain and double chain t-PA were purified.

For example, Bowes melanoma cell culture medium (
10% heat-inactivated (56°C, 30 minutes) fetal bovine serum and 2
Twee 2ff (containing 0KIU/ml of abrotin)
After stabilization with n80 (0.02%) and common salt (0.4 mol/l), it was applied to a 5TI-Sepharose column.

The effluent was collected and measured for plasminogen-dependent fibrinolytic activity, which was approximately 98% of the activity applied to the column.
% was confirmed. Add salt to both parts at a final concentration of 1.0 mol/
After stabilization, it was applied to an ETI-Sepharose column.

The effluent was collected and plasminogen-dependent fibrinolytic activity was measured. Approximately 10% of the activity applied to the column was confirmed. When these two components were examined by zymography after SOS polyacrylamide gel electrophoresis, the blasminogen activation was found to be 110,000 ± 20,000 Daltons and 7
Two types of 1,000,000 daltons were confirmed. After passing the entire solution through an ETI-Sepharose column, the column was washed with 20 column volumes of 1.0 M saline containing 0.2% Tween 80. By this method, approximately 5% of the activity applied to the column was detected, and bands of 110,000±20,000 and 70,000 daltons were identified as plasminogen activators on the zymograph.

The adsorbed protein was dissolved in 0.2
Elution was carried out by a near-gradient method from pH 5.s to pH 3.0 using M citrate buffer.

This method gave one peak in the range from pl+5.2 to pl+4.5 and another peak in the range from pH 4.5 to p)13.7. These two fractions together gave an activity of 80-85x applied to the column.

As a result of examining the sample reduced with mercaptoethanol by staining after polyacrylamide gel electrophoresis, PH5,
The molecular weight of those eluted in the range from pH 2 to pH 4.5 did not change even after reduction and showed 70,000 Daltons, but at pH 4.
, 5 to pl (3,7). When reduced, the 70,000 Dalton band disappeared and a band around 30,000 to 40,000 Daltons was confirmed. From this result, pl (5
. It was confirmed that it was p^.

Example 2 Human fetal foreskin cell culture solution [10% 0% heat inactivated 6°C, 30
) containing fetal bovine serum and 20 KIU/ml of aprotin) with Tween 80 (0,02%) and saline (
After stabilization at 0.4 mol/β), it was passed through a 5TI-Sepharose column.

The effluent was collected and plasminogen-dependent fibrinolytic activity was measured. Approximately 98% of the activity applied to the column was confirmed. Salt was added to both portions to give a final concentration of 1.0 mol/1 for stabilization, and then applied to an ETI-Sepharose column.

The effluent was collected and plasminogen-dependent fibrinolytic activity was measured. Approximately 45% of the activity applied to the column was confirmed. When these two components were examined by zymography after SDS polyacrylamide gel electrophoresis, it was found that 2 to 3
Two to three bands were confirmed around 50,000 to 70,000 Daltons, and one band was found around 35,000 Daltons.

1. For washing the ETr-Sepharose column that adsorbed t-FA. 0.20 column volumes containing OM NaCl and 0.2χ Tween 80. IM NHJC (h PH7
The column was washed with the buffer solution of .

Approximately 5% of the activity applied to the column was detected by this method and the same bands as above were confirmed on the zymograph.

The only difference from Example 1 was that the elution buffer was O, LM glycine-HCl buffer containing 0.15 M NaCl (pl(4,5) and pH 3,5). was carried out in exactly the same manner, and the following results were obtained.

A combination of the two separated fractions with different pH values showed an activity of 40-50x applied to the column. When the reagent reduced with mercaptoethanol was examined by silver staining after polyacrylamide gel electrophoresis, the molecular weight of those eluted at pH 4 and 5 did not change even after reduction and showed 70,000 daltons, but P) 13 When the sample eluted at .5 was reduced, the band at 70,000 Daltons disappeared and a band at around 30,000 to 40,000 Daltons was confirmed.

From this result, it was confirmed that the t-PA eluted at pH 4.s was single-stranded t-PA, and the t-PA eluted at pH 3.s was double-stranded -PA.

Example 3 A culture solution of mouse fibroblasts incorporating the human t-PA gene [containing 2% heat-inactivated (56°C, 30 minutes) fetal bovine serum and 20 KIU/ml abrotin] 2j2 was added to Tw
een80 (0.02%) and salt (0.4 mol/1
) was applied to a 5TT-Sepharose column.

The effluent was collected and plasminogen-dependent fibrinolytic activity was measured. Approximately 98% of the activity applied to the column was confirmed. After stabilization, common salt was added to both portions to give a final concentration of 1.0 mol/l, and the mixture was applied to an ETr-Sepharose column.

The effluent was collected and plasminogen-dependent fibrinolytic activity was measured. Approximately 10% of the activity applied to the column was confirmed. When these two components were examined by imography after SOS polyacrylamide gel electrotransfer, it was found that the plasminogen activator was 110,000 ± 20,000 Daltons and 7
Two types of 1,000,000 daltons were confirmed. After passing the entire solution through an ETr-Sepharose column, the column was washed with 20 column volumes of 2.0 M saline containing 0.2 χ Tween 80. By this method, approximately 5% of the activity applied to the column was detected, and bands of 110,000±20,000 and 70,000 daltons were confirmed as plasminogen activation on the zymograph.

The adsorbed protein contains 0.2
Eluted with M sodium phosphate solution pH 4,5 and pl (3,5).

Together these two fractions represented approximately 80% of the activity applied to the column. When the reagent reduced with mercaptoethanol was examined by silver staining after polyacrylamide gel electrophoresis, the molecular weight of the one eluted at pH 4 and 5 did not change even after reduction and showed 70,000 daltons, but the When the eluted product was reduced, the band at 70,000 Daltons disappeared and a band around 30,000 to 40,000 Daltons was confirmed. From this result, t-PA eluted at pH 4 and 5 is single-chain t-P.
A, and it was confirmed that the t-PA eluted at pl+3.5 was double-stranded t-PA.

Patent applicant: Mitsui Toatsu Kagaku Co., Ltd. Procedural Amendment (sealed) August 6, 1988 Director General of the Patent Office Kunio Ogawa 1, Indication of the case: 1985 Patent Application No. 103200 2, Title of the invention: 1 Method 3 of separating strand t-PA and double strand t-p^, relationship with the case of the person making the amendment Patent applicant address 3-2-5 Kasumigaseki, Chiyoda-ku, Tokyo Name (31
2) Mitsui Toatsu Chemical Co., Ltd. 5, Claims column and Detailed Description of the Invention column 6 of the specification to be amended, Contents of the amendment (1) The scope of claims of the specification as attached. to correct.

(2) In the specification, page 2, lines 6 to 8, “We are t-
Knowing that PA activity...is omitted...is composed of two chains," is corrected to J as single-chain tPA and double-chain tPA exist among substances with rtp^ activity.

(3) Similarly, on page 2, line 13, change “There is.” to “
(D, C, Rijken et al + J, B
oil, Che+n, 256 +7035~7041
．． 1981). ” he corrected.

(4) Similarly, on page 2, line 20, “Known.

” [known (Bio-Pooi catalog (S
wede-n). ” he corrected.

(5) Similarly, “more than 80%” on page 3, line 14
Delete.

(6) Similarly, on page 4, lines 10-11, "human normal cells" is corrected to "human normal cells."

(7) Similarly, on page 4, lines 12-13, “-heavy chain,
Double-stranded t-PAJ is defined as “-heavy chain t-PA and double-stranded t-PA
Correct it with J.

(8) Similarly, on page 4, line 15, "Ippon product" is corrected to "Ippon 41t-pA."

(9) Similarly, page 4, line 17 [Hereafter
This will be explained using PA as an example. ” [Hereinafter, the present invention will be specifically explained with reference to Examples. ” he corrected.

00) Similarly, on page 5, line 4, "processed."
Processed (Joubert et al+Hoppe-
5eyler's Z, P-hysiol, chem,
, 362.531-538.1981), corrected as J.

(11) Similarly, on page 5, lines 7 and 8, "centrifuge the extract...process omitted...recover from the supernatant" is changed to "
Salt out the supernatant with ammonium sulfate, collect the precipitate, and correct it as j.

(121Same as 126mH on page 5, line 15'
Corrected to 25mgJ.

Attachment 2, Claims 1) - Terminated tPA and double stranded tPA are stored ↓ Once the ETI has been carried, the end chain tPA and the double strand tPA are stored.
Single chain tPA and double chain tPA characterized by these tPA
How to separate PA.

"Hiro"

Claims (1)

[Claims] A mixture containing single-chain t-PA and double-chain t-PA is first passed through a column carrying ETI.
Single-stranded t-PA and double-stranded t-PA, which are characterized by retaining PA and then eluting these t-PAs with a solution that is more acidic and a solution that is more alkaline than pH 4.5. Method for separating t-PA.