JPH03160990A - Method for activating insoluble proteins produced with genetically modified bacteria - Google Patents

Abstract

PURPOSE:To sterilize a protein without causing the denaturation and decomposition of the objective protein by carrying out the heat-treatment of a fraction containing the insoluble protein, addition of a reducing agent and the subsequent removing operation prior to the activation operation of the insoluble protein produced by genetic engineering process and accumulated in cells. CONSTITUTION:An insoluble protein is produced by a gene recombination microorganism and accumulated in the microbial cell. The protein is activated by disintegrating the cell, recovering the insoluble fraction, suspending the recovered component and activating in a state capable of expressing the activity. The above process is carried out as follows. The fraction containing the protein is subjected to heat-treatment and addition of a reducing agent at an arbitrary time from the disintegration of the cell to the activation operation and the added reducing agent is removed from the above fraction after the heat- treatment and the addition of the reducing agent and before the activation operation. The reducing agent is e.g. 2-mercaptoethanol or bithioerythritol. The heat-treatment is carried out e.g. at about 70-90 deg.C for about 0.5-1hr for the sterilization of E.coli.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for activating an insoluble protein produced by a genetically modified bacterium prepared by genetic engineering techniques.

(Prior art) With recent advances in genetic engineering technology, target proteins, peptides, etc. (hereinafter simply referred to as proteins unless otherwise specified) can be produced using bacteria such as Escherichia coli, Bacillus subtilis, or yeast. It is now possible to produce it within the body. The protein of interest is often a so-called hetero-8 protein that is not naturally produced by the bacterial cells used as the host.

In particular, when the produced target proteins are heterologous proteins, they may be present in the host as insoluble proteins (insoluble aggregates).
fusion bodies) and may accumulate. The target protein that has formed an insoluble mass is folded into a form (tertiary structure) that cannot express its original physiological activity, so it is necessary to perform an activation operation called solubilization (unfolding) and refolding ( As the activation operation, for example, JP-A-59-161321, JP-A-60-5
No. 00893 is known).

By the way, when handling organisms manufactured by genetic engineering methods or microorganisms into which foreign genes have been incorporated, it is important to prevent biohazards, and for this reason, heat treatment, heat treatment, etc. It is common to carry out sterilization treatments such as acid treatment, alkali treatment or organic solvent treatment.

(Problems with the Prior Art) When a heterologous protein is produced by a bacterial cell, the protein is often accumulated in the bacterial cell as an insoluble protein. It becomes necessary to crush the body, collect an insoluble fraction, suspend the fraction, and perform an activation operation.

In such operations, heat treatment, acid treatment, and alkali treatment are necessary from the viewpoint of preventing the above-mentioned biological hazards or contamination of protein preparations that are ultimately activated into physiologically active forms by bacterial cells. Alternatively, it is desirable to carry out sterilization treatment such as organic solvent treatment, but when these treatments are carried out, the produced protein itself is denatured or decomposed, resulting in a problem that the amount of recovered protein is reduced.

(Means for Solving the Problems) In view of the above-mentioned problems of the prior art, the present inventors particularly performed sterilization treatment by heating during a series of operations to obtain insoluble proteins in a form capable of expressing activity. After careful consideration of the method of carrying out the heat treatment, we found that by carrying out the heat treatment with the target protein in contact with the reducing agent, or by bringing the target protein into contact with the reducing agent after the heat treatment, the target protein would not be denatured or decomposed. discovered that it is possible to sterilize
The present invention has been completed. That is, the present invention deals with insoluble proteins produced by genetically modified bacteria and accumulated in the cells, by disrupting the cells, collecting the insoluble fraction, suspending it, and activating it into a form capable of expressing activity. In a series of operations, the fraction containing the protein is subjected to heat treatment and a reducing agent is added at any point from crushing the bacterial cells to activation; This is a method for activating an insoluble protein produced by a genetically modified bacterium, which comprises performing an operation to remove the added reducing agent from the fraction after and before the activation operation. The present invention will be explained in detail below.

The present invention relates to a series of operations for obtaining a target protein in a form capable of expressing its original physiological activity, using bacterial cells containing the target protein as an insoluble mass.

Therefore, there are no restrictions on the bacterial cells themselves, as long as they are microorganisms used in common genetic engineering methods, such as Escherichia coli, Bacillus subtilis, or yeast, and there are no restrictions on the target protein.

In the present invention, the fraction containing insoluble proteins refers to the bacterial cells themselves that have accumulated insoluble fractions, a solution containing the bacterial cells, a bacterial cell suspension obtained by disrupting bacterial cells, or a fraction recovered from the bacterial cell disruption solution. This refers to a fraction that mainly contains the protein of interest, such as an insoluble fraction. In the present invention, known reducing agents such as 2-mercaptoethanol, dithiothreitol, and dithioerythritol can be used. The amount of these reducing agents to be added may be appropriately determined in consideration of the temperature of the heat treatment to be carried out and its lIs, and there are no particular limitations. As will be explained later in the Examples of the present invention, to give an example of the amount added in the treatment of E. coli that has accumulated prourokinase, the final concentration of these reducing agents in the fraction containing insoluble protein is By setting the content to 0.6% or more, it is possible to obtain the effect of preventing protein denaturation and decomposition due to heat treatment, but preferably 0.05% or more.
The effect can be further enhanced by setting it to 5% or more.

The heat treatment carried out in the present invention may be carried out by appropriately determining the time and temperature necessary to sterilize the bacterial cells. For example, for E. coli, the treatment may be carried out at 70 to 90°C for 0.5 to 1 hour.

In the present invention, there are no restrictions on the heat treatment method itself, and there are no restrictions on the operations attached to the heat treatment, but in order to prevent the target protein from denaturation and decomposition6, it is necessary to contain insoluble protein after the heat treatment. It is best to cool the fraction.

The reducing agent may be added before the heat treatment, after the heat treatment, or even at the same time as the heat treatment, but the reducing agent used may be added during the heat treatment. If the material is easily affected by heat treatment, it is preferable to add it after heat treatment. The added reducing agent is separated prior to activation of the insoluble protein of interest. This prevents disulfide bonds from being poorly formed in the refolding operation in the presence of a reducing agent, which prevents a good activation operation from being achieved. This operation itself can be easily accomplished by operations such as centrifugation and ultrafiltration, taking advantage of the fact that proteins are insoluble before being solubilized in the activation operation.

The present invention will be specifically explained below.

Operation for disrupting one microbial cell The microbial cells may be disrupted by a physical method or a chemical method, and examples of the physical method include homogenization and a method using ultrasound. Examples of chemical methods include adding lysozyme or an appropriate surfactant, but these methods tend to cause irreversible denaturation of the target protein, so physical methods are used and the method is carried out in a solution. It is preferable to do so.

As explained above, the present invention can also be implemented in the crushing operation of bacterial cells. In other words, after collecting the bacterial cells from the culture medium by centrifugation or other operations, suspending them in an appropriate buffer, adding a reducing agent, heat treatment, and then crushing the bacterial cells. good. Moreover, it is also possible to carry out the invention at the time when bacterial cell disruption is completed.

2. Recovery of insoluble fraction from crushed bacterial cell solution The insoluble fraction may be recovered by centrifugation, ultrafiltration, or the like on a suspension of crushed bacterial cells. When carrying out the present invention using the obtained insoluble fraction, the obtained fraction may be suspended in an appropriate buffer or the like.

3 Activation Operation The activation operation usually consists of a treatment to solubilize an insoluble protein and a treatment to refold the solubilized protein.

During refolding, if the target protein and reducing agent coexist, the formation of correct disulfide bonds will be hindered, so the reducing agent added prior to this operation is separated. This is because after the solubilization treatment, the target protein exists as a soluble protein, making it difficult to separate the reducing agent. As an activation operation, for example, Japanese Patent Application Laid-Open No. 591613
No. 21, JP-A No. 60-500893, etc. are known, but the present invention is not particularly limited thereto.

(Operations of the Invention) According to the present invention, in a series of processing operations for obtaining an insoluble protein produced by bacterial cells in a form capable of expressing physiological activity, bacteria can It is possible to sterilize the body and reduce the possibility of causing a biohazard or contaminating the final prepared protein with the bacteria. Furthermore, the method of the present invention can be achieved by relatively simple operations of adding a reducing agent and performing heat treatment, and can be implemented within existing equipment for genetic engineering techniques.

In the present invention, sterilization can be achieved by a physical method called heat treatment, so there is no need to use alkaline substances, acidic substances, surfactants, or other chemicals, and the cost is low. There is no need to worry about contamination of the target protein.

Furthermore, according to the present invention, it is possible to inactivate protease derived from bacterial cells that may degrade target proteins, and as a result, the yield of target proteins can be increased.

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

The bacterial cell used in this example was Escherichia coli (KY1436 strain), and the target protein produced was human brourokinase (hereinafter referred to as prourokinase). Prourokinase is a foreign protein to E. coli and accumulates within E. coli as an insoluble protein.

The activity of the produced pro-urokinase was determined by treating pro-urokinase with protease and converting it into urokinase, using pyroglutamyl, glycyl, and arginyl paranitroanilide hydrochloride, which is a specific substrate of urokinase. method (Biotechn
2, 628, 1984), and the activity derived from the insoluble fraction per 1 g of wet bacterial cells when no superheating treatment or addition of a reducing agent was performed.
Relatively expressed as %.

Example 1 Escherichia coli was transformed with a 11 plasmid containing the gene encoding prourokinase and cultured in M9 medium (the plasmid was described in JP-A-62-143686). (Deposited as No. 8341 with the National Institute of Microbial Technology on July 11, 1985). When the OD of the medium reaches 10, add I to the medium.
Protein expression was induced by adding PTG, and the cells were further cultured for 7 hours.

After the culture was completed, the culture solution containing the bacterial cells was crushed using a homogenizer (manufactured by Manton-Gorlin) to obtain a solution of crushed bacterial cells. The solution was centrifuged and an insoluble fraction containing the produced prourokinase was collected. Suspend the insoluble fraction equivalent to 1 g of wet bacterial cells in 10 ml of pure water, and incubate at 70°C for 1 hour.
After heat treatment for an hour, the final concentration was 0.5.
% or 20 mM.

After standing at room temperature for 30 minutes, centrifugation was performed to collect the insoluble fraction.

Subsequently, the collected insoluble fraction was
Activation 1 2 was performed according to the method described in No. 1. First, the insoluble fraction was suspended in 20 ml of 0.1 M Lis-HCl buffer (pH 8.0), and then 20 ml of 8 M
After adding the guanidine hydrochloride solution and leaving it at room temperature for 2 hours, 0.05M} Lis-HCl buffer (pH 8.0) was added to lower the guanidine hydrochloride concentration to IM, and then further 0.2
Glutathione was added to a concentration of mM and left overnight.

Table 1 shows the prourokinase activity in the obtained solution. In addition, in the table, ME represents 2-mercaptoethanol and DTT represents dithiothreitol.

1 3 Table 1 According to Table 1, there is only a slight change in urokinase activity depending on the presence or absence of the addition of a reducing agent when heat treatment is not performed, but there is a large difference when heat treatment is performed; It can be seen that denaturation and arcing are prevented.

1 4 Example 2 An insoluble fraction equivalent to 1 g of wet bacterial cells prepared in the same manner as in Example 1 was suspended in 10 mM phosphate buffer (pH 7.0) containing 5 mM EDTA, and incubated at 70°C for 30 minutes or 1
After heating for a period of time, the mixture was cooled with water, and a portion thereof was collected to measure the number of surviving bacterial cells and the protease activity derived from the bacterial cells. In addition, 2-mercaptoethanol was added to the remaining solution so that the final concentration was 0.5%, and 3-mercaptoethanol was added at room temperature.
After standing for 0 minutes, centrifugation was performed to collect the insoluble fraction.

The recovered insoluble fraction was subjected to the same activation procedure as in Example 1, and then its prourokinase activity was measured. The results are shown in Table 2.

The number of surviving bacterial cells was determined from the number of colonies produced by culturing the collected solution on an M9 agar medium at 30° C. for 3 days, and the number of bacterial cells per ml of the cultured solution. The protease activity derived from bacterial cells was determined by reacting the collected solution with 1% casein (pH 7.5) at 37°C, stopping the reaction with 1-lichloroacetic acid, and then measuring the absorbance at 280 nm of the acid-soluble fraction. The relative activity was calculated based on the activity derived from the insoluble fraction corresponding to 1 g of wet bacterial cells that had not been heat treated as 100%.

Table 2 From Table 2, heat treatment at 70°C for 30 minutes is sufficient to sterilize E. coli, and in that case, protease activity derived from the bacterial cells is reduced by about half, but prourokinase activity is reduced by more than 80%. It can be seen that 1 6 remains. In this way, heat treatment has the effect of sterilizing living cells and also reduces the activity of protease derived from bacterial cells that may degrade the target protein.Additionally, the addition of a reducing agent denatures and degrades the target protein. It can be seen that it has the characteristic that it is prevented.

Example 3 The insoluble fraction equivalent to 1 g of wet bacterial cells obtained in the same manner as in Example 1 was added to 10 mM phosphate buffer (p
H7.0) and at a temperature of 80°C or higher for 30 minutes or 1
After heat treatment for an hour, cool on ice until the final concentration is 0.05.
2-mercaptoethanol was added to the mixture to give a concentration of ~1.0%, the mixture was left to stand at room temperature for 30 minutes, and the insoluble fraction was collected by centrifugation.

The collected insoluble fraction was subjected to the same activation procedure as in Example 1, and the prourokinase activity a1 was determined.

The results are shown in Table 3. In addition, ME in the table is 2-Me1 7 This indicates lucaptoethanol.

1 8 From Table 3, when prourokinase is produced using Escherichia coli, if it is heat treated at 80°C for 1 hour or 90°C for 30 minutes, the addition of 0.5% 2-mercaptoethanol will reduce the concentration by about 50%. It was found that the activity of
Even in heat treatment at 90℃ for 1 hour or 30 minutes in a boiling state, by adding about 0.5% reducing agent,
It can be seen that ~30% of the activity is protected.

Example 4 An insoluble fraction equivalent to 1 g of wet bacterial cells obtained in the same manner as in Example 1 was suspended in pure water, treated at 70°C for 30 minutes, cooled with water, and centrifuged to remove the insoluble fraction. Recovered.

Regarding prourokinase in the collected insoluble fraction,
The activity was measured as described above.

As the reducing agent, 2-mercaptoethanol was added at a concentration of 0.5% before the heat treatment or 15 minutes after the start of the heat treatment (during the heat treatment). The results are shown in Table 4.

1. From Table 4, it can be seen that the reducing agent may be added before the heat treatment (see the results in Table 1) or during the heat treatment.

Claims (1)

[Claims] (1) Regarding insoluble proteins produced by genetically modified bacteria and accumulated in the bacterial cells, a series of steps are taken to crush the bacterial cells, collect the insoluble fraction, suspend it, and activate it into a form capable of expressing activity. In the operation, the fraction containing the protein is heat treated and a reducing agent is added at any point from the crushing of the bacterial cells to the activation operation, and after the heat treatment and the reducing agent is added. 1. A method for activating an insoluble protein produced using a genetically modified bacterium, which comprises performing an operation for removing the added reducing agent from the fraction before the activation operation.