JPH0451893A - Method for recovering urokinase-like enzymic precursor - Google Patents

Abstract

PURPOSE:To simply improve the yield by culturing a specific microorganism and then performing crushing operation in the presence of a metallic chelating agent without separating the microorganism from a culture medium. CONSTITUTION:A strain of Escherichia coli KY1436, etc., is transformed with a plasmid capable of coding a urokinase-like enzymic precursor to provide a transformed Escherichia coli (A), which is then cultured in a culture medium containing glycerol and casein, etc., to afford a culture solution (B). A metallic chelating agent such as EDTA in an amount of 5-100mM is subsequently added to the aforementioned culture solution (B). The resultant culture solution, together with the microbial cells, is treated at pH7-10 in a homogenizer to provide a suspension (C) of the crushed Escherichia coli. The obtained ingredient (C) is subsequently treated in a centrifugal separator, etc., and converted into an insoluble fraction to recover the urokinase-like enzymic precursor (D). The resultant ingredient (D), as necessary, is dissolved in guanidine hydrochloride, etc., then subjected to reactivating operation such as dialysis or purification to produce the objective urokinase-like enzymic precursor.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention applies to E. coli transformed with a plasmid containing a gene encoding a urokinase-like zymogen. The term urokinase-like zymogen used herein refers to, for example, Japanese Patent Application Laid-Open No. 59-5130.
In addition to the natural type urokinase precursor described in No. 0, etc., it also includes those in which a part of the amino acid sequence is substituted with an amino acid different from the natural one (Japanese Patent Application Laid-open No. 1438813/1983, etc.). ).

The medium referred to in the present invention refers to the medium in which E. coli that is actually subjected to the crushing treatment is cultured, that is, the medium itself in which E. coli that has accumulated urokinase-like enzyme precursors in its cells is suspended in the final stage of culture. It means something.

As the metal chelating agent used in the present invention, for example, EDTA, EGTA, etc. may be used.

These may be added directly to the above-mentioned medium.

Although there is no particular restriction on the concentration used, the effects of the present invention can be sufficiently achieved by using about 5-100 mM.

After adding the metal chelating agent, E. coli can be disrupted together with the culture solution in the medium using, for example, a homogenizer, an ultrasonic disruptor, or the like. At this time, the pH of the medium is preferably maintained within the range of pH 7 to 10, so that the urokinase-like enzyme precursor is not inactivated. Therefore, measure the pH of the medium,
Trishydroxylaminomethane/hydrochloric acid as needed,
The pH may be maintained within the above range by adding phosphate, glycine, caustic soda, or the like.

After the crushing operation is completed, the urokinase-like enzyme precursor is recovered as an insoluble fraction, but in the present invention, this operation is not limited, and may be performed by, for example, centrifugation, ultrafiltration, or the like.

As a result of the above operation, when subjected to the reactivation operation as explained later, the urokinase-like enzyme precursor recovered from E. coli simply suspended in the medium and subjected to the disruption operation is Urokinase-like enzyme precursors in a state capable of expressing high activity can be recovered.

On the other hand, the present invention provides a recovery method in which the urokinase-like enzyme precursor recovered as described above is then subjected to a reactivation operation to recover a specimen in a state capable of expressing activity. be. Regarding the reactivation operation, for example, the urokinase-like enzyme precursor is solubilized in a solution of a strong protein denaturant such as guanidine hydrochloride, and the concentration of the denaturant is then lowered by diluting or dialysis. (see JP-A-59-161321) and pH 10-1.
The urokinase-like enzyme precursor is solubilized in an alkaline aqueous solution of about 3%, and then an acidic substance is added to adjust the pH of the solution.
A method of lowering the value (see Japanese Unexamined Patent Publication No. 130-500893) may be used.

(Effect of the invention) When the cells are disrupted in the medium and the urokinase-like enzyme precursor is recovered and the reactivation operation is performed without separating E. coli from the medium, the urokinase-like enzyme precursor with low activity is The problem arises that only the body can be recovered. If this problem were to be solved, for example, by centrifugation, it would be necessary to install large-scale equipment or to perform a reactivation operation after separating E. coli and the medium over a long period of time. According to the invention, similar effects can be achieved without such large-scale equipment or lengthy processing. That is, the present invention performs a crushing operation without separating the bacterial cells from the medium by an extremely simple operation of adding a metal chelating agent to the medium,
The present invention provides a method for recovering urokinase precursor-like protein in which the yield of the urokinase precursor-like protein does not decrease even if a subsequent activation operation is performed. In detail, as shown in the Examples below, by adding a metal chelating agent to the medium, urokinase-like cells that have high activity even when the subsequent reactivation procedure is performed without separating E. coli from the medium. This makes it possible to obtain enzyme precursors.

(Examples) Examples will be described below to explain the present invention in more detail, but the present invention is not limited to these Examples.

Furthermore, since the urokinase-like zymogen itself does not express any activity (because it is an zymogen), its activity is determined by converting it into a urokinase-like enzyme by treating it with plasmin (P
yrGlyArg-pNA; manufactured by Daiichi Chemical Co., Ltd.)
The hydrolytic activity of Hayas
hj S, Thromb, Res, Volume 22, p573
-578.1981).

Example 1 ``N'' described in JP-A-62-143 [i86]
A plasmid encoding a urokinase-like enzyme precursor in which the amino acids at positions 135 and 157 counting from the end are replaced with lysine and phenylalanine, respectively.Escherichia coli KY1
43B strain was transformed and cultured in M9 medium containing glycerin and casein decomposition products until Illoon turbidity reached 50.

The following operations were performed on lft of the above-mentioned media. In addition, 50 g of bacterial cells (wet weight) were contained in 1 g of the culture medium.

First, the pH of the medium was adjusted to about 9 by adding 12.1 g of trishydroxyaminomethane (Tris) and hydrochloric acid, and the pH of the medium was adjusted to about 9.
11.9 g of ethylenediaminetetraacetic acid (EDTA) was added. Next, this medium containing 50 g of bacterial cells was subjected to a homogenizer (Gorlin homogenizer), and
The bacterial cells were disrupted using PS and centrifuged at 8000 rpm for 20 minutes to collect the insoluble fraction. Subsequently, the collected insoluble fraction was suspended in 500+all of O, 1M Tris-HCl buffer (pH 8.0), and 500a+47 of 8M
After adding the guanidine hydrochloride solution, it was left to stand at 40°C for 2 hours to solubilize. After completing the solubilization treatment, S mM
EDTA, 0.2 mM reduced glutathione, 0.0
6fI of 50IIIM Tris-HCl buffer (pH 8,0) containing 2mM oxidized glutathione was added to carry out reactivation treatment.

When the obtained urokinase-like enzyme precursor was treated with plasmin as described above, the total amount of the urokinase-like enzyme precursor was about 135
5000. OIJ activity was recovered. In this example, 5
Since 0g of wet bacteria was used, 1g of wet bacteria was 27
This means that 1000 U of activity was recovered.

Comparative Example 1 The following comparative example was carried out using 1 g of the culture medium obtained in Example 1. Note that, as in Example 1, approximately 500 g (wet weight) of bacterial cells was contained in 1 g of the medium.

12゜1g trishydroxyaminomethane (Tris)
and hydrochloric acid to adjust the pH of the medium to approximately 9, and then the medium containing 50 g of bacterial cells was subjected to a homogenizer (Gorlin homogenizer) to crush the bacterial cells at 8000 ps.
Centrifugation was performed for 20 minutes at 8000 rpffl to collect the insoluble fraction. Subsequently, the collected insoluble fraction was
0.114 of 00ml) Liss-HCl buffer (pH 8,
After adding 500-8M guanidine hydrochloride solution, the mixture was left to stand at 40°C for 2 hours to solubilize. After completing the solubilization process, 5 mM EDTA.

5011M Tris-HCl buffer (pH
8,0) was added to carry out reactivation treatment.

When the obtained urokinase-like enzyme precursor was treated with plasmin as described above, the total amount of the urokinase-like enzyme precursor was approximately 855%.
0000 tl of activity was recovered. In this comparative example, 50
Since 1g of wet bacteria was used, 1g of wet bacteria was 17
Only 1000 U of activity, or 67% of Example 1, was recovered.

Comparative Example 2 The following comparative example was carried out using 11) of the culture media obtained in Example 1. In addition, the 1f! As in Example 1, the medium contained approximately 500 g (wet weight) of bacterial cells.

First, the culture medium containing this bacterial cell was heated at 8000 rpm for 2 hours.
Centrifugation was performed for 0 minutes to collect 50 g of solids.

This bacterial cell was dissolved in 500 J of Tris-HCl buffer (1) H8.0.
), use a homogenizer (Gorlin homogenizer) to crush the bacterial cells at 8,000 ps,
Centrifugation was performed at rpm for 20 minutes to collect the insoluble fraction. Subsequently, the collected insoluble fraction was added to 50 tnl of 01
Suspended in M Tris-HCl buffer (1) I (8,0),
After adding 8M guanidine hydrochloride solution of 4
It was left to stand at 0°C for 2 hours to solubilize. After completing the solubilization treatment, 5mM containing 5mM EDTA, 0.2mM reduced glutathione, and 0.02mM oxidized glutathione was added.
6g of 0mM Tris-HCl buffer (pH 8,0) was added to carry out reactivation treatment.

When the obtained urokinase-like enzyme precursor was treated with plasmin as described above, the total concentration of the urokinase-like enzyme precursor was approximately 126
50000 U of activity was recovered. In this comparative example, 50
Since I used 1g of wet bacteria, 1g of wet bacteria has a strength of 253
000 U of activity, that is, 94% of the activity of Example 1 was recovered.

Claims (2)

[Claims] (1) After culturing E. coli transformed with a plasmid containing a gene encoding a urokinase-like enzyme precursor in a medium, disruption is performed in the presence of a metal chelating agent without separating the E. coli from the medium. A method for recovering urokinase-like enzyme precursor by subjecting it to water and recovering the insoluble fraction. (2) After culturing E. coli transformed with a plasmid containing a gene encoding a urokinase-like enzyme precursor in a medium, disruption is performed in the presence of a metal chelating agent without separating the E. coli from the medium. A method for recovering urokinase-like enzyme precursors, in which the insoluble fraction is recovered by subjecting the enzyme to urokinase, followed by reactivation.