JPH05502795A - Method for purifying proteins present in inclusion bodies - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for Purifying Proteins Present in Inclusion Bodies The present invention purifies proteins in the form of insoluble inclusion bodies. This invention relates to a method for purifying proteins produced by A.

The present invention particularly relates to proteins produced in Escherichia Co11. , particularly regarding methods for purifying proteins produced by genetic engineering. .

DNA recombination technology has been used in numerous ways in Escherichia Co11. Expression of atypical proteins can occur. These proteins are produced in large quantities However, it is produced in insoluble form. In practice, these proteins are called inclusion bodies. It is precipitated in the form of aggregates that are exposed.

Thus, for example, human growth hormone, bovine growth hormone, interferon Elon, interleukin 2, and plasminoken activators are produced.

In particular, the gene encoding brourokinase (pr o-UK) is Esch Clonin in Erichia Co11 (EP 365.894) was produced and expressed.

pro-UK is the single chain natural form of urokinase.

Urokinase is a serine compound known for its thrombolytic and fibrinolytic properties. It is an enzyme of the protease group.

Therefore, urokinase has important clinical uses as a thrombolytic agent.

However, urokinase is systemic in its action and is commonly used in treatment. At high doses, there is a risk of bleeding.

As is known, the fibrinolytic enzyme system depends on plasminokene, Ken is activated and becomes plasmin, which catalyzes the degradation of plasmin or fibrin. Ru.

Alpha 2 Plasmin inhibitors such as Antiplasmin and Plasminoken A distinction is made between active agents. between plasminoken, its active agent and fibrin. Coordination is carried out through mutual molecular interactions. Of the plasminoken activators, double-chain Urokinase has a strong affinity for plasminokene and the absence of fibrin It also activates plasminoken. This is the difference from brourokinase brourokinase has a similarly strong affinity for plasminokene. However, it appears that plasminoken is activated only in the presence of fibrin. and thus exhibit relative specificity for fibrin clots. This kind of whole-body activity Because of the absence of sex, the use of brourokinase is particularly effective in therapy.

Initially, brourokinase was isolated from human urine and various cell cultures. However, in order to obtain the recombinant protein, the expressed gene was isolated and cloned. It is now possible to produce green products.

Especially recombinant proteins by bacteria in E+cheNchia Co11 production involves purification problems.

In effect, the recombinant protein is secreted inside insoluble inclusion bodies. Traditionally, the culture medium Concentrate the microbial cells by centrifugation or ultrafiltration. mechanically rupture cell membranes Disrupt by trituration, sonication, or enzymatic treatment, then centrifuge the inclusion bodies Collected by. During this centrifugation, the supernatant liquid contains soluble components of bacteria. Contains. However, the particulate fraction collected in this way, i.e. the precipitated material In addition to inclusion bodies containing recombinant proteins, unbroken cells, cell debris and Contains soluble or insoluble protein contaminants.

The coarse precipitated material is then washed away. This cleaning is a gentle process to remove soluble contaminants. It is carried out with an aqueous solution.

However, this process involves additional mixing and centrifugation steps and is easy to implement on a large scale. do not have.

Next, insoluble proteins present in the precipitated material are denatured by changes in the medium composition. Dissolve by action. That is, in U.S. Pat. No. 4,694,073 recombinant tumatropin by addition of urea and/or dimethylsulfone is dissolved. U.S. Pat. Solutions containing anidine, urea, SDS or T+1jon are proposed. Next Insoluble components must be removed by centrifugation, which results in incomplete ligation. produce fruit.

The protein is then renatured by inhibition of denaturants in the medium. This is the solution This is done by diluting the liquid.

A post-purification step is then performed by ion-exchange chromatography or gel filtration. implement.

Therefore, the present invention is directed to proteins secreted by bacteria in the form of insoluble inclusion bodies. a) in the form of a precipitated material by disruption and centrifugation of microbial cells; collecting the inclusion bodies; b) suspending the precipitated material with a solution of a water-soluble material capable of being gelled; C) The mixture of said precipitated material and said aqueous material solution is added to a gelling solution, e.g. Dropped into a solution containing cations to encapsulate the precipitated material within the gelled material. d) at least one chaotropic agent and optionally a reducing agent. placing the encapsulated precipitated material into a lysis solution containing; e) obtaining a denatured and purified protein in solution. It is.

variants of some proteins, especially those whose genes are introduced into bacterial genomes Proteins are ripened by this bacterium into inclusion bodies. These inclusion bodies are , distinguished by their refractive index, form insoluble aggregates in the cytoplasm.

In order to purify proteins, it is first necessary to separate inclusion bodies. Therefore, Pre-concentrated microbial cells by centrifugation, microfiltration or ultrafiltration Perform mechanical crushing.

After crushing, the precipitated material is separated by centrifugation at 4,000 to 5,000 g. Suspend in buffer solution.

As a buffer solution, in particular T+is-HCI lOm! J, pH 8.5 is used. Ru. All of these steps were carried out before 4°C to inhibit proteases present during the preparation. Perform at a later temperature.

Suspension of a precipitated substance in a solution of a water-soluble substance that can be gelled is a solution of this water-soluble substance. is carried out by mixing with a suspension of precipitated material. This process also It can also be carried out by adding water-soluble substances such as to the precipitated material suspension.

A water-soluble substance that can be gelled is defined as a water-soluble substance that can be used to form a gelling solution, e.g. a solution exhibiting a constant pH. Retains an essentially solid droplet shape upon exposure to polyvalent cations or by exposure to polyvalent cations It means a substance that can produce an object.

Among these substances, natural polysaccharide gums such as pectin, and alkali metal Mention may be made of alginates. Preferred materials are alginates, especially alginate. It is sodium chloride.

These alginates of alkali metals are particularly sensitive to the presence of polyvalent cations. By dropping into solutions containing valent cations, especially calcium chloride solutions. gelled.

Alkali metal ions are ion-exchanged by multivalent ions to form a gel, resulting in precipitated substances. form a capsule. This capsule is a reticulated gel that encloses insoluble compounds. It consists of However, this capsule is permeable to liquid substances.

Therefore, the next step consists in denaturing the protein present in insoluble form. the Add a lysis solution to the encapsulated precipitated material. This lysis solution is at least Contains one type of chaotropic agent, whose function is to modify the spatial shape or conformation of proteins. It consists in changing the homeation and causing its dissociation. In addition, the dissociation solution At least one reducing agent that cleaves the quality disulfide bridges can be included.

As chaotropic agents, mention may be made of urea and guanidine hydrochloride. good A preferred chaotropic agent is 6M guanidine hydrochloride◎ Reducing agents include 2-mercaptoethanol and dithiothreitol. I can do things. A preferred reducing agent is 53mM 2-mercaptoethanol. child Due to the action of the dissociation solution, the denatured proteins become soluble and the encapsulated particles freed from the child. Proteins exit the capsule, but insoluble contaminants remain inside the capsule. It remains in storage. The capsule must be resistant to denaturing media.

In the next step the protein must be renatured. Therefore, insoluble After particle removal, the solution is gradually diluted by the addition of a buffer solution. Therefore, the denaturant concentration The protein is renatured in high yield. Protein recovery rate is 100% Close to.

Then, by ion exchange chromatography and hydrophobic chromatography Perform final purification of the protein.

This technique is reproducible and can easily be extrapolated to large scale. This technology Eliminates the need for a centrifugation step of the solution. This is because the removal of insoluble compounds from capsules This is because it is carried out through hoarding.

The encapsulation of precipitated material facilitates its cleaning. Accordingly, the present invention provides for carrying out the dissolution step. before applying, the encapsulated precipitated material is washed with a buffer solution. This relates to the purification process described above. Preferably 0.1% Triton Using a 10mM, pH 8.5 buffer solution containing Tri+-HCl, The precipitated material contained in the solution is kept warm at low temperature (4-6°C) with gentle stirring.

This step allows soluble compounds to be removed and encapsulated precipitated material to be removed from the buffer solution. After being suspended in a liquid, it is placed in a lysis solution.

The coarse precipitated material obtained after disruption and centrifugation of microbial cells is washed before storage. Ru. The present invention also provides a method for purifying secreted proteins in the form of inclusion bodies. The present invention relates to a method for washing and homogenizing a sample before suspending it. In particular, precipitated substances are 011 10 mM, p[I 8.5 buffer solution TriS-H containing % Triton It can be cleaned with CI. The washed precipitated material is removed by microfiltration. Concentrated and suspended in 10mM, pti 8.5 buffer solution Tri+-HCl. It becomes cloudy.

The present invention provides that the protein is secreted by the bacterial species E+cherichia Co11. The present invention relates to a method characterized by:

In fact, it is difficult to select a species of Cucilia that is particularly suitable for protein production. did it. These proteins can be secreted in the form of inclusion bodies.

E+ cherichia Co11 is the host of choice for gene cloning.

Therefore, the present invention provides that the recombinant tan I flesh is Escberichia Co11. A method for purifying a protein characterized by being a recombinant protein produced by It is related to.

Escberichia Co11 by vectors that function in bacteria Genes for various proteins can be introduced into the cells.

In particular, the present invention provides a protein secreted by Escberichia Co11. A protein characterized in that the quality thereof is recombinant burourokinase (pr o-UK). This relates to a method for purifying protein.

The table below shows the mode of implementation for the optimal purification and renaturation of brourokinase.

Centrifugation (precipitated material) 30 1000 4 Microfiltration 5 500 2 concentrated overstage Due to the dissolution solution consisting of − next dilution The restoration efficiency of brourokinase is as follows: Sea urchin treated with plasmin and then treated with L-pyroglutamyl-glycyl-al Confirmed by measuring urokinase activity on ginine-p-nitroanilide substrate. determined. In this case, plasmin is inhibited by aprotinin. Brad Weigh the protein by Ford's reagent.

Example Precipitated material encapsulation in calcium alginate granules A sample of the precipitated material used was 0. lomM, pH 8.5 buffer solution Tris-HC containing 1% Taiwan Washed twice with I.

15ml of precipitated material was mixed with 2.5mt of 10mM, pH 8.5 buffer solution T+i+- Mix with 17.5 g of sodium alginate at 40 g/l in HCl and water.

This mixture was then poured into 200 ml of lOQff 1M calcium chloride solution at 4°C. Drip into.

A quantity of particles corresponding to 5 ml of precipitated material was intermittently added to 125 ml of standard lysis solution. Incubate at 4°C for 18 hours with gentle stirring. This solution was prepared using 6M hydrochloric acid glycol. Contains anidine and 50mM 2-mercaptoethanol.

After reconstitution, the activity of the obtained brourokinase is measured.

A standard lysis process is performed on control precipitated material without encapsulation.

The results obtained are shown in Table 1.

Table 1 *IU: L-pGL[1-GLY-ARG-p-++ troanilide (5 -2444, Kabi4im+m, Sweden) at 37°C and pH 8.8. min, the amount of enzyme that releases 0.1275 nanomoles of nitroaniline.

Example 2 Encapsulation of precipitated material in calcium alginate gel, washing of encapsulated precipitated material, Lysing and restoring rheuurokinase Insoluble materials (cells, cell fragments, inclusion bodies, etc.) are contained within the calcium alginate gel. easily stored. Performs simple washing of encapsulated precipitated material instead of several steps of centrifugation. This method was used to illustrate the possibility of applying Especially when using guanidine hydrochloride. It must be noted that special cleaning procedures can easily be introduced if used.

- 10 g of precipitated material specimens (protein content , 60%) at 4°C (10 minutes, 10.000 rpm). The resulting separator was immersed in lomM containing 0.1% TrNon, pH 8.5. Washed twice with buffer solution T+1s-HCI. The obtained washed product was treated with 0.1% Suspended in the same buffer containing and without TrNon (final suspension volume: 10 g) .

- Encapsulation of precipitated material and washing of encapsulated precipitated material 10 g of precipitated material (unwashed; protein content (260%) mixed with 3.3 g of 4% calcium alginate solution do. The encapsulation operation was performed as previously described.

The encapsulated precipitated material was then dissolved in 10 mL pH 8.5 containing 0.1% Triton. Washed once with buffer solution T+1s-1 (CI).

The washed suspension was mixed with a solution containing guanidine hydrochloride and a reducing agent and solution 5.7. The mixture was mixed at a ratio of 1 g of initial precipitated material sample to 5 mf.

In this way, for 1 g of the initial precipitate material sample, the volume of the dissolution solution is 6.85 ml for substance and 5.4 ml for encapsulated precipitated material.

Table 2a: Cleaning table for encapsulated precipitated substances and non-encapsulated precipitated substances 2b Composition of restoration solution Table 2C: Protein recovery and whether precipitated material is included or not. Comparison of pro-UK activity These results demonstrate that the precipitate in calcium alginate gel Precipitate retention not only retains undissolved compounds during dissolution operations, but also This shows the advantage that precipitated substances can be efficiently washed without the need for heart separation operations.

Example 3: Dissolution of precipitated material by use of sequential lofts of guanidine hydrochloride Add 5 ml of the suspension of precipitated material to 2.5 μl of 10 mM, pH 8.5 buffer solution. Mix with Tti+-HCl. Add 7.5 ml of this solution to 4% (p/v) Algium Sodium phosphate SG-500 (Me+o-Roassetot-3atia, Made in France).

The mixture of sodium alginate and precipitated material was heated to 100 mM calcium chloride at 4°C. into the solution.

After gently stirring the particles or spheres in the calcium chloride solution for 15 minutes, The encapsulated precipitated material was washed twice with IQmM, pH 8.5 buffer solution Tr-HCl. Wash.

The encapsulated precipitated material is introduced into 125 ml of standard lysis solution.

After the first dissolution period of 9 hours at 20°C, the guanidine hydrochloride solution was removed and the same conditions The second lysis lot will be carried out.

The activity and specific activity of pro-UK are shown in Table 3.

pro-U recovered after encapsulating precipitated material in calcium alginate particles The total amount of K units is much higher than pro-UK recovered by standard method (+ 180%). The specific activity of this sample is three times that of the standard solution.

Electrophoresis (5DS-PAGE) of the first lysis lot was performed using the standard lysis method. The band corresponding to a molecular weight of 30 kD is shown with lower intensity than that obtained.

Table 3 6M guar hydrochloride after encapsulating the precipitated material in calcium alginate gel Dissolve brourokinase in ginseng and 50mM 2-mercaptoethanol. Test standard method Example 4= pr using calcium alginate particles in the column.

-UK dissolution method 7 ml of the precipitate suspension was mixed with 2.5 ml of 10 mM, pH 8.5 buffer solution T+i. Mix with s-[ICt. This solution was added to 7 ml of 4% (p/v) alginate at 4°C. Mix with Sodium Acid 5G-500. The procedure in this case is the same as in Example 3. It is.

Calcium alginate particles were introduced into a 1.5×9 cm column. this color Standard lysis solution was applied at a flow rate of 22 ml/hour for the first 6 hours and then for the next 6 hours. , at a flow rate of 5 ml/hour. This test was conducted at 4°C.

In the eluent, the absorbance at 280 mn is continuously measured. Protein content was tracked.

The results are shown in Tables 4 and 5.

Due to the low diffusion rate between gel and lysis solution (Table 5), calcium alginate Only 50% of the protein is eluted from the lamb.

Table 4: After encapsulating the precipitated material in calcium alginate gel, 6M hydrochloric acid Dissolve prourokinabi in anidine and 50mM 2-mercaptoethanol. Contains protein by volume, activity, total activity, total protein, specific activity. fraction ml U/ml U mg/ml mg υ/Q1g1 22 126 0 693000 Q, lO5512600230143010720000, 11821300032212106650000, lQ 55 121004 22 890 490000 0, IG 55 89005 22 8H48 40000,1055880061111903270000,1233990 071011102770000, 15377400811110030300 00,12339200 table 5. of precipitated material in calcium alginate gel Recovery solution of pro-UK after encapsulation and dissolution of pro-UK in fixed bed column Volume of solution pro-UK activity protein specific activity test 150 4.3 11.000 89 405 51 10.640 Required book The present invention describes a method for purifying proteins secreted by bacteria in the form of insoluble inclusion bodies. It is about law. According to this method, (a) inclusion bodies are formed by disrupting microbial cells; (b) The precipitated material is gelled. (C) encapsulating the precipitated substance in the gelled substance; (d) the mixture of precipitated material and water-soluble material is dropped into the gelling solution; The encapsulated precipitated material contains at least one chaotropic agent and optionally (e) denatured and purified tan; ( A solid solution is obtained.

international search report III 1 - 1T "w11+-噌-i--Po^寥TN-口" ie, PCT/FR9+-100629

Claims (12)

[Claims] 1. A method for purifying proteins secreted by bacteria in the form of insoluble inclusion bodies. There, a) collecting the inclusion bodies in the form of precipitated material by disruption of the microbial cells and centrifugation; and, b) suspending the precipitated material with a solution of a water-soluble material capable of being gelled; c) The mixture of said precipitated material and said aqueous storage solution is dissolved in a gelling solution, e.g. Dropped into a solution containing cations to encapsulate the precipitated material within the gelled material. d) at least one chaotropic agent and optionally a reducing agent. placing the encapsulated precipitated material into a lysis solution containing; e) obtaining a denatured and purified protein in solution form. Method. 2. Before carrying out said dissolution step (e), the encapsulated precipitated material is washed with a buffer solution. 2. The purification method according to claim 1, further comprising the step of washing by washing. 3. Claim characterized in that before carrying out suspension step b), the precipitated material is washed. The purification method according to either 1 or 2. 4. The gelatinable water-soluble substance is sodium alginate. The dilution method according to any one of claims 1 to 3, wherein: 5. 5. The method according to claim 1, wherein the gelling solution contains polyvalent cations. The purification method described in any of the above. 6. 6. The method according to claim 1, wherein the gelling solution is a calcium chloride solution. The purification method described in any of the above. 7. The chaotropic agent is selected from urea and guanidine hydrochloride. The purification method according to any one of claims 1 to 6. 8. Claim 1, wherein the reducing compound is 2-mercaptoethanol. 8. The purification method according to any one of 7. 9. The protein is secreted from the bacterial species Escherichia coli. The purification method according to any one of claims 1 to 8, characterized in that: 10. Claim characterized in that the secreted protein is a recombinant protein. 9. 11. Claim characterized in that the secreted protein is pro-urokinase. Item 11. The purification method according to item 9 or 10. 12. Introducing encapsulated precipitated material into a fixed bed column before entering the lysis solution The purification method according to any one of claims 1 to 11, characterized in that: