MXPA01001168A - Process for the preparation of active somatotropin from inclusion bodies - Google Patents

Abstract

A process for the preparation of biologically active somatotropin from inclusion bodies of a recombinant host cell containing an inactive form of said somatotropin protein comprises the steps of:(a) contacting the inclusion bodies with an aqueous alcohol solution at an alkaline pH to solubilize said protein;and (b) bringing the solubilized protein into contact with a mild oxidizing agent to refold and form intramolecular disulfide bonds between cysteine residues of said protein.

Description

PROCESS FOR THE PREPARATION OF SOMATOTROPIN ACTIVE FROM INCLUSION BODIES FIELD OF THE INVENTION The present invention relates to a process for the preparation of somatotropin from inclusion bodies. More specifically, it belongs to a process for the preparation of the somatotropin, biologically active, from inclusion bodies of the recombinant host cell, which contains the somatotropin protein, which comprises solubilizing the inclusion bodies with an aqueous alcoholic solution and redouble and oxidize the solubilized somatotropin protein to deliver the active somatotropin.

BACKGROUND OF THE INVENTION Heterologous proteins, expressed in transformed host cells,. they occur either in a soluble form or in the form of inclusion bodies, inactive and insoluble (or refractive bodies) depending on such factors as the redox environment of the host cell, the level of expression and the nature of the protein. There is also a case of limit line, in which both soluble and insoluble forms of proteins are expressed simultaneously.

Proteins expressed in the form of inclusion bodies must be solubilized and renatured to obtain biologically active proteins, and several methods have been developed for the renaturation of proteins produced in the form of inclusion bodies. A common method uses a high concentration of denaturing chaotropic agents, for example the urea and guanidine salts, in solubilizing the bodies and the solubilized proteins are renatured after dilution or ultrafiltration of the solution to decrease the concentration of the denaturing agent . In this method, the solubilization process is carried out at a high concentration, for example from 5 to 9 M, of the denaturing agent and the renaturation process, at a low concentration, for example from 1 to 5 M (see, for example, example, European patent No. 0 114 506). In addition, it has been reported that the addition of a small amount of an alcohol to the solution containing the denaturing agent increases the rates of solubilization and redoubling (see USP 5,109,117). A second method carries out the solubilization of the inclusion bodies at a high pH, in which a protein is denatured, adding an alkali, such as NaOH, to a solubilizing solution and renaturing the protein by lowering the pH (see, for example , Korean Patent No. 86911). This method can be effectively combined with the common method, mentioned above, to unleash the strong non-covalent attractions between the proteins in the inclusion bodies. A third method employs a surfactant for the solubilization of the inclusion bodies (see, for example, U.S. Patent No. 5,023,323). The hydrophobic portion of the surfactant dissipates the attractive force between the proteins in the inclusion bodies by interacting strongly with the proteins, while its hydrophilic portion is in close contact with the water. Thus, the surfactant solubilizes the inclusion bodies by self-binding to the protein monomers in an aqueous solution, to form a stable structure. However, this method has the problem that it is difficult to remove the surfactant, which interacts strongly, from the renatured proteins. Protein inclusion bodies, which have isoelectric points generally at an approximately neutral pH, are formed when the protein monomers do not have net charge aggregates at a neutral pH, due to the strong non-covalent attractions, among them. A denaturing agent, when present at a higher concentration, displaces the non-covalent attractions, thus solubilizing the inclusion bodies. In addition, at a highly alkaline pH, the amino acid residues of proteins become charged, thus facilitating the solubilization of the inclusion bodies. When a protein, which has cysteine residues, is placed at a high enough pH for the dissociation of the free SH groups, from the cysteine residues, oxidation by a moderate oxidant, such as air, occurs slowly, to form intramolecular disulfide bonds, between the cysteine groups. However, these intermolecular disulfide bonds can also be formed between two or more protein monomers, which result in the formation of diamonds, trimers and other polymeric forms of the protein. In fact, known methods for renaturation of somatotropin are hampered by the problem that the proportion of the dimeric and polymeric forms of somatotropin is relatively high and, therefore, there is a need to develop an effective method to produce somatotropin. biologically active with high performance, while minimizing the formation of the dimeric and polymeric forms of somatotropin. As it is difficult to remove the inactive dimeric and polymeric forms of the somatotropin, in a purification process, it is important to suppress its formation in the processes of redoubling and reoxidation. The present inventors have discovered, unexpectedly, that the use of a concentrated alcohol solution, which does not contain chaotropic agents, particularly an aqueous solution of isopropyl alcohol or n-propyl alcohol, in the solubilization process, significantly reduces the formation of other forms. dimeric and polymeric somatotropin.

SUMMARY OF THE INVENTION Therefore, it is a primary object of the present invention to provide an improved process for the renaturation of somatotropin. In accordance with the present invention, a process for the preparation of the biologically active somatotropin is provided, from the inclusion cords of a recombinant host cell, which contains an inactive form of said somatotropin protein, this process comprises the steps of : (a) contacting the inclusion bodies with an aqueous alcohol solution at an alkaline pH, to solubilize the protein; and (b) contacting the solubilized protein with a moderate oxidation agent, to redouble and form intramolecular disulfide bonds, between the cysteine residues of said protein.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which: Figure 1 shows the correlation between the concentration of the n-propyl alcohol, in the solubilizing step, and the solubility of the somatotropin, as determined by the reductive electrophoresis of the polyacrylamide gel of sodium dodecyl sulfate (SDS-PAGE); Figure 2 illustrates the correlation between the concentration of isopropyl alcohol in the solubilization step and the solubility of the somatotropin, as determined by the reducing SDS-PAGE; Figure 3 shows the correlation between the concentration of isopropyl alcohol in the step of redoubling / reoxidation and the renaturation regimen of the somatotropin, as determined by the non-reducing SDS-PAGE; Figure 4 shows the change in the solubility of the somatotropin as a function of the temperature in the solubilization step, as determined by the reducing SDS-PAGE, Figure 5 shows the renaturing regime of the somatotropin, as a function of the concentration of 2-mercaptoethanol, in the step of redoubling / reoxidation, as determined by the reductive / non-reducing SDS-PAGE; and Figure 6 shows the solubility of the somatotropin as a func tion of the concentration of the somatotropin in the solubilization step, as determined by the reductive / non-reducing SDS-PAGE.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the following terms will have the meanings as described below: The term "somatotropin" refers to the growth protein hormones of mammals, birds or fish. It may include, but is not limited to, the somatotropin of humans, cattle, pigs, horses, goats, sheep, canines, felines, chickens, turbot fish, rockfish, salmon or eels. In addition, it may also include somatotropin analogues in which the amino acid residues are or are deleted from, added to, or different from, the naturally occurring amino acid sequences of somatotropins, so long as the analogues maintain the bioactivities of those that they occur naturally. For example, the present invention can be used for the naturalization of bovine somatotropin (BST), and porcine somatotropin analogues (PST), including ala-BST, met-BST, ala-PST and met-PST. It is well known in the field of protein purification that proteins having analogous physico-chemical properties can be purified according to substantially the same procedures and, therefore, the present invention also includes the purification of the analogous proteins of proteins. the somatotropin. The term "inclusion bodies" refers to the cytoplasmic aggregates containing the heterologous proteins expressed in the transformed host cell, which can be recovered by the cytoplasm separation. These aggregates look like bright areas under a microscope. The term "transformed host cell" refers to a microbial cell, which contains a heterologous gene to express a somatotropin. Exemplary host cells include prokaryotic cells, such as an E. coli cell, as well as eukaryotic cells, such as a yeast cell. The term "solubilization" refers to the dissociation of the inclusion bodies into unitary protein molecules by an appropriate treatment. The suspension, which contains the inclusion bodies, becomes clear as the solubilization progresses. The term "redoubled" refers to the folding of the molecules of dissociated proteins, produced in the process of solubilization in their native three-dimensional conformation. This procedure is carried out by the amino acid sequence of the protein. It is well known that disulfide bonds are formed in the correct positions when the redoubling precedes the formation of disulfide bonds in a protein, thus causing the formation of an active protein of the native conformation. The term "reoxidation" refers to the formation of intramolecular disulfide bonds, between the cysteine residues of the redoubled protein molecules, through an oxidation reaction. Since the redox potential in an E. coli cell is high, the cysteine residues of the somatotropin exist as a reduced state and, therefore, a stable somatotropin molecule can be formed, only when the reoxidation process is it subsequently carries out the solubilization process for the stabilization of the three-dimensional structure. The present invention employs a high concentration of water-soluble alcohol, preferably isopropyl alcohol or n-propyl alcohol, for the solubilization of the somatotropin produced in the form of inclusion bodies. These alcohols are believed to change the polarity of the water to weaken the non-covalent attraction in the inclusion bodies, and to displace the non-covalent attractions between the protein molecules. If the pH of the solution in the solubilization process increases to a highly alkaline value, the amino acid residues of the protein become charged to further weaken the non-covalent attraction between the proteins. Next, the solubilized somatotropin is contacted with a moderate oxidizing agent, for example oxygen or hydrogen peroxide, in the presence of the alcohol used in the solubilization process, preferably at a concentration lower than that used in the process of solubilization, to prepare the active somatotropin, through the process of redoubling / reoxidation. The general process for purifying the somatotropin of a recombinant E. coli cell, using the present invention, can be illustrated as follows. The inclusion bodies, which contain the somatotropin, can be recovered by any conventional method, for example, that described in Korean patent No. 86911. Specifically, the E. coli cells expressing the somatotropin are altered with a homogenizer, in a cell disruption regulator, which contains a nonionic surfactant and insoluble inclusion bodies are recovered using a centrifuge. The recovered inclusion bodies are washed with a regulator, which contains a nonionic surfactant and ethyl n-diamine tetraacetic acid (EDTA) and, subsequently, with water, to remove the impurities. The washed inclusion bodies are suspended in an aqueous solution of alcohol, preferably 10 to 50% (volume / volume) of a solution of isopropyl or n-propyl alcohol, more preferably, a solution of 20 to 40% isopropyl alcohol. , at a concentration of 1 to 10 grams / liter. The suspension is maintained at a temperature ranging from 0 to 50 ° C, preferably from 30 to 50 ° C, more preferably from 35 to 40 ° C, and at a pH greater than 9, preferably from 12 to 13, and then allowed to stand for a period of time that varies from 5 to 30 minutes, to dissolve the inclusion bodies. Subsequently, the resulting solution is diluted with water or subjected to ultrafiltration to decrease the alcohol concentration to 1 to 35%, preferably 10 to 20%, when the alcohol is isopropyl or n-propyl alcohol. The solution is then exposed to air and stirred moderately for 1 to 8 hours, to redouble and effect the oxidative formation of the intramolecular disulfide bonds between the cysteine residues of the somatotropin protein, thus producing the active somatotropin. The redoubling / reoxidation reaction is carried out at a temperature ranging from 0 to 50 ° C, preferably from 5 to 40 ° C, more preferably from 10 to 20 ° C, and at a pH greater than 8.4, preferably from 12 to 13. At this time, a metal catalyst, for example CuCl 2, MgSO 4 and CaSO 4, can be added to the reaction mixture to promote the reaction. In addition, the redoubling / reoxidation reaction can also be carried out after the removal of isopropyl alcohol or n-propyl alcohol. In the present invention, a reducing agent can be added to the reaction mixture in the solubilization process, the redoubled / re-oxidation process or both of them, in order to increase the rate of formation of the correct disulfide bonds. In the case of the solubilization process, from 0.0001 to 0.5% of 2-mercaptoethanol or a correspondingly effective amount of another reducing agent, for example cysteamine, glutathione, cysteine and their redox mixtures, can be used, and it is preferred to employ 0.001 0.05% (volume / volume) of 2-mercaptoethanol. In the case of the redoubled / reoxidation process, from 0 to 0.25% of the 2-mercaptoethanol or a correspondingly effective amount of other reducing agents, exemplified above, can preferably be employed. Upon completion of the renaturation of the somatotropin, the activated somatotropin can be further purified according to a conventional method, for example, that of Korean patent No. 86911, which includes ultrafiltration and anion exchange chromatography, to obtain the somatotropin. pure with high performance. The renaturation process of the invention is advantageous in that it is possible to obtain an active somatotropin with high yield from the inclusion bodies produced in a recombinant host cell, without employing a denaturing agent, for example urea, or a surfactant agent, for example sodium dodecylsulfate (SDS), which is conventionally employed. The following examples further attempt to illustrate the present invention, without limiting its scope. The percentages given below for the solids in the mixture of solids, liquids in liquids, and solids in liquids are based on weight / weight, volume / volume and weight / volume, respectively, unless specifically indicated otherwise.

Example 1: Effect of Addition of n-Propyl Alcohol in the Solubilization Process (Stage 1) Recovery of Inclusion Bodies Recombinant E. coli cells expressing bovine somatotropin (CFC-10693) deposited on May 25 of 1990, with the Korean Federation of Culture Collection (Korean Federation of Crop Collections) (Address: College of Engineering, Yonsei University, Sodaemun-Gu, Seoul 120-749, Republic of Korea)), were cultured in a 450 liter fermenter and a 300 liter aliquot was adjusted to a pH of 4.8 by the addition of acetic acid. The resulting solution was centrifuged with a continuous centrifuge (BTPX 2150, Alpha-Laval) to recover an aqueous paste of E. coli cells. The aqueous paste was suspended in distilled water at a final volume of 200 liters, with the addition of 0.1% Triton X-100 and 10 mM EDTA. The suspension was passed twice through a high pressure homogenizer (SHL 15, Alpha-Laval) at 980 kg / cm 2 or a flow rate of 200 liters / hour for cell disruption, and the resulting homogenate was subjected to continuous centrifugation to obtain an aqueous paste containing the inclusion bodies. The aqueous paste was suspended in 120 liters of an aqueous solution containing 0.625% Triton X-100 and 50 mM EDTA. The suspension was shaken vigorously with Polytron (PT 10/35, KINEMATICA GMBH) for 30 minutes, diluted with distilled water to 240 liters and centrifuged to obtain precipitates. These precipitates were suspended in 180 liters of distilled water, shaken vigorously with Polytron (PT 10/35, KINEMATICA GMBH) for 15 minutes, and centrifuged to obtain inclusion bodies as precipitates. The precipitates were suspended in 100 liters of distilled water.

(Step 2) Solubilization of inclusion bodies and redoubling / reoxidation of bovine somatotropin To each of five 350 ml portions of the inclusion body suspension obtained in Step 1, 100, 200, 300 were added, 400 or 500 ml of n-propyl alcohol and distilled water was added to a volume of 1 liter. The n-propyl alcohol concentrations of these suspensions correspond to 10, 20, 30, 40 and 50% (volume / volume), respectively. The temperature of each suspension was adjusted to 37 ° C, an aqueous solution of NaOH was added, with moderate agitation, at a pH ranging from 12 to 13, and then allowed to stand for 10 minutes, to completely dissolve the inclusion bodies . The resulting solubilized solution was diluted with distilled water to 2 liters, stirred moderately under air at room temperature, for 2 hrs, and then centrifuged. The resulting supernatant and the pellets were analyzed with the reducing SDS-PAGE, to determine the contents of the somatotropin. The result is shown in Figure 1, where strips 1 to 5 represent, respectively, the supernatants obtained using 10, 20, 30, 40 and 50% (volume / volume) of the n-propyl alcohol in the solubilization process; and strips 6 to 10, the corresponding precipitates. As can be seen in Figure 1, the amount of undissolved somatotropin remaining in the precipitates begins to decrease in strip 7 (20% (volume / volume) of n-propyl alcohol) and remains almost constant at a low level in strips 8 (30% n-propyl alcohol) to 10 (50% n-propyl alcohol). This result shows that the somatotropin was effectively solubilized in an aqueous solution containing 20% or more of the n-propyl alcohol.

Example 2: Effect of Adding Alcohol Isopropyl in the Solubilization Process In order to investigate the effect of the addition of isopropyl alcohol on the solubilization of the somatotropin, the procedure of Example 1 was repeated, except that the isopropyl alcohol was used in the solubilization process, instead of n-propyl alcohol. Figure 2 shows the result of reducing the SDS-PAGE, in which strips 1 to 5 correspond, respectively, to the supernatants obtained using 10, 20, 30, 40 and 50% (volume / volume) of isopropyl alcohol in the solubilization process; and strips 6 to 10, the corresponding precipitates. As can be seen in Figure 2, the amount of the undissolved somatotropin remaining in the precipitates is significantly low in the strip 8 (30 &volume 7 volume of isopropyl alcohol), and remains approximately constant at a low level in strips 9 (40% isopropyl alcohol) and 10 (50% isopropyl alcohol).

Example 3: Effect of Varying the Concentration of Isopropyl Alcohol in the Redoubled / Reoxidation Process In order to investigate the effect of varying the concentration of isopropyl alcohol in the renaturation of the somatotropin, the procedure of Example 1 was repeated, except that 350 ml (35%) of the isopropyl alcohol were added in the solubilization process. The solubilized solution, thus obtained, was used as such in the redoubled / re-oxidation process or diluted with distilled water to a final volume of 2 liters or 4 liters, before being subjected to the redoubled / re-oxidation process. As can be seen from the result of the non-reducing SDS-PAGE in the Figure, the amount of the renatured somatotropin is greater when the solubilized solution was diluted 2 times (strip 2) than the undiluted case (strip 1 | or when diluted 4 times (strip 3).

Example 4: Effect of Temperature in the Case Solubilizer In order to investigate the effect of varying the temperature in the solubilization of the somatotropin, the procedure of Example 1 was repeated, except that 350 ml (35%) of the isopropyl alcohol were added and the temperature varied from, 10, 15, 20, 25, 30, 35, 40, 45 to 50 ° C, in the solubilization process. The solubility (%) of the somatotropin was determined with the reducing SDS-PAGE and the result is shown in Figure 4. As shown in Figure 4, a maximum regime above 30 ° C was observed.

Example 5 Effect of adding a Reducing Agent (2-mercaptoethanol) in the Redoubling / Re-Oxidation Process In order to investigate the effect of adding the 2-mercaptoethanol in the redoubling and reoxidation of the somatotropin, the procedure of Example 1 was repeated, except that 350 ml (35%) of the isopropyl alcohol were added in the solubilization process and the 2-mercaptoethanol was added as a reducing agent, at a concentration of O, O.OOl, 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.08 or 0.1% in the solubilizing process. The renaturation rate (%) of the somatotropin was determined with the reductive / non-reducing SDS-PAGE and the result in Figure 5 shows that the renaturation regimes are 62, 73, 77, 79, 80, 81, 82, 74, 64 and 45%, respectively.

Example 6 Effect of Adding a Reducing Agent (Cysteamine) in the Redoubling / Reoxidation Process In order to investigate the effect of adding the cysteamine in the redoubling and re-oxidation of the somatotropin, the procedure of Example 1 was repeated, except that 350 ml (35%) of isopropyl alcohol was added in the solubilization process, 5 mM cysteamine was added as the reducing agent in the redoubled / re-oxidation process and the reaction mixture was stirred for 6 hours. The renaturation regimen (%) of the somatotropin, determined with the reductive / non-reducing SDS-PAGE, was 78%.

Example 7: Effect of Varying the Concentration of Somatotropin in the Solubilization Process In order to investigate the effect of varying the concentration of the somatotropin in its solubilization, the procedure of Example 1 was repeated, except that 350 ml (35%) of isopropyl alcohol were added and the concentration of bovine somatotropin in the suspension of the inclusion body was adjusted to 0.85, 0.95, 1.0, 1.2, 1.4, 1.7, 2.1, 2.9, 4.3 or 8.8 grams / liter in the solubilization process . The solubility (%) of the somatotropin was determined by the reducing SDS-PAGE (Quantitation and comparison of the amounts of the somatotropin in the supernatant and precipitates) and the result in Figure 6 shows that the extension of the solubilization varies from 70 , 75, 82, 88, 89, 92, 88, 95, 86 to 53%, is in accordance with the change in the concentration of the somatotropin, as before.Example 8: Purification of Somatotropin (from Bovine, Expressed in E. coli.

(Stage 1) Recovery of inclusion bodies Recombinant E. coli cells expressing bovine somatotropin (KCTC 0644BP) deposited on July 2, 1999 in the Korean Collection for Type Cultures (Korean Collection for Type Cultures) (Address: Korea Research Institute of Bioscience and Biotechnology, # 52, Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea), under the terms of the Budapest Treaty, in the International Recognition of the Deposit of Microorganisms, for the Purposes of the Patent Procedure), were subjected to a batch culture in a 10 liter tank and the pH of the resulting culture was adjusted to 4.8 by the addition of acetic acid. The culture ("Cell F") was centrifuged with continuous centrifugation (BTPX 2150, Alpha-Laval) to collect pellets from E. coli cells. The pellets were suspended in distilled water at a final volume of 4 liters, with the addition of 0.1% Triton X-100 and 10 mM EDTA. The suspension ("S cell") was passed twice through a microfluidizer (Microfluidics Corporations) at 560 kg / cm2 for the disruption of the cells, and the resulting homogenate ("Cell H") obtained in a yield of 91.2% of Cell F, was subjected to continuous centrifugation, to deliver precipitates containing the inclusion bodies. The precipitates were suspended in 10 liters of an aqueous solution containing 0.625% Triton X-100 and 50 mM EDTA. The suspension was shaken vigorously with a Polytron (PT 10/35, KINEMATICA GMBH) for 30 minutes, diluted with distilled water to 5 liters and centrifuged to obtain precipitates in a yield of 47.2%, based on the weight of the cell. H. The precipitates were diluted with distilled water to 2 liters, and the resulting suspension ("TEW") was stirred vigorously with Polytron for 15 minutes and centrifuged (centrifuge at high speed J2-21M BECKMAN) to obtain precipitates with a yield of 81.4%, based on the TEW. The precipitates were suspended in distilled water at 0.5 liter and the resulting suspension ("WFIW-1") was stirred and centrifuged as before, to obtain precipitates in a yield of 89.6%, based on WFIW-1. The precipitates were suspended in distilled water at 0.5 liter.

(Stage 2) Solubilization of inclusion bodies and redoubling / reoxidation of bovine somatotropin 250 ml of the suspension of the inclusion bodies (WFIW-2") obtained in Step 1 was added to 350 ml (35% ) of isopropyl alcohol and 0.02% of 2-mercaptoethanol, and the mixture was adjusted to 1 liter by the addition of distilled water.The concentration of the bovine somatotropin in the resulting suspension was approximately 2.5 grams / liter. heated to 37 ° C and its pH adjusted to 12-13 with the addition of an aqueous NaOH solution with moderate agitation, and then allowed to stand for 10 minutes, to completely dissolve the inclusion bodies. distilled water to 2 liters, and stirred moderately under air, at room temperature, for 2 hours.

(Step) Purification of bovine somatotropin The solution of the inclusion bodies, obtained in Stage 2, was centrifuged to remove the insoluble impurities and the supernatant was subjected to ultrafiltration to obtain a filtrate containing the materials with a molecular weight below of 300,000 Daltons. The filtrate was subjected to ultrafiltration to obtain a retentate containing substances with a molecular weight greater than 10,000 Daltons. The retentate was diluted with water for injection (WFI) and then passed through an ultrafiltration membrane (cut-off molecular weight: 10,000 Daltons) to obtain a concentrate. This dilution and concentration process was repeated four times and the concentrate was diluted with distilled water to obtain a solution having a conductivity of 800 micro-mho and a pH of 9.0. This solution contained the somatotropin with a yield of approximately 100%, based on the amount of the somatotropin in WFIW-2. 2 liters of the resulting solution ("LS") were adsorbed on a DEAE-Sepharose column (Pharmacia Biotech), at a flow rate of 20 liters / hour and sufficiently washed with 10 mM of a glycine regulator. The adsorbed material was diluted with a glycine buffer containing 90 mM NaCl to obtain fractions containing the bovine somatotropin ("DEAE"). The overall yield of the purified bovine somatotropin was finally about 28.5%, based on the amount of the somatotropin in the F cell.

Comparative Example 1: Renaturation of the Bovine Somatotropin, Which Uses a Denaturing Agent (Urea) The renaturation of bovine somatotropin was performed according to Holzman's method (T. F.

Holzman et al., Biochemistry, 25, 6907-6917 (1986)).

Specifically, 250 ml of the suspension of the inclusion body, obtained in Step 1 of Example 8, were diluted with distilled water to a final volume of 1.3 liters, with the addition of urea at a concentration of 4.5 M.

The resulting solution was adjusted to a pH of 10.5 by the addition of an aqueous NaOH solution and stirred at room temperature for 8 hours. The bovine somatotropin was purified from the resulting solution, according to the method of Step 3 of Example 8. The yield of renaturation (%) of the bovine somatotropin in the step of redoubling the urea was 83.7%, based on the amount of the somatotropin in WFRW-2 and the overall purification yield was 20.4%, based on the amount of the somatotropin in the F cell.

Example 8: Purification of Porcine Somatotropin, Expressed in E. coli Inclusion bodies, containing porcine somatotropin, were recovered, according to the method of Step 1 of Example 8, from the E. cells. recombinant coli, expressing porcine somatotropin (KCTC 0523BP; deposited on September 24, 1998, at the Korea Research Institute for Type Cultures Institute (Address: KCTC, KRIBB, # 52, On-dong, Yusong-ku, Taejon 305 -333, Republic of Korea) under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures), and the procedures of Steps 2 and 3 of Example 8 were repeated. The concentration of porcine somatotropin in the suspension of the inclusion body in the solubilization process was 2.3 grams / liter. The yield of renaturation (%) of porcine somatotropin was approximately 90%, based on the amount of the somatotropin in the WFIW-2 and the overall purification yield was 28.5%, based on the amount of somatotropin in Cell F.

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes in the invention can be made by those skilled in the art, which are also within the scope of the invention, as defined by the attached claims.

Claims (19)

1. CLAIMS 1. A process for the preparation of the somatotropin, biologically active, from inclusion bodies of a recombinant host cell, containing an inactive form of said somatotropin protein, this process comprises the steps of: (a) putting contacting the inclusion bodies with an aqueous solution of alcohol, at an alkaline pH, to solubilize said protein; and (b) bringing the solubilized protein into contact with a moderate oxidizing agent, for redoubling and forming intramolecular disulfide bonds between the cysteine residues of said protein.
2. 2. The process of claim 1, wherein the alcohol is n-propyl alcohol or isopropyl alcohol.
3. 3. The process of claim 2, wherein the concentration of the isopropyl alcohol or the n-propyl alcohol, in step (a), ranges from 10 to 50%.
4. 4. The process of claim 2, wherein the concentration of the isopropyl alcohol or the n-propyl alcohol, in step (b), ranges from 10 to 50%.
5. 5. The process of claim 2, wherein the isopropyl alcohol or the n-propyl alcohol are removed before step (b).
6. 6. The process of claim 1, wherein the moderate oxidizing agent is air.
7. 7. The process of claim 1, wherein the recombinant host is E. coli.
8. 8. The process of claim 1, wherein said somatotropin is from a mammal, bird or fish.
9. 9. The process of claim 1, wherein said somatotropin is human, bovine, porcine, horse, goat, sheep, canine, feline, chicken, turbot, rockfish or salmon fish, or of the angula.
10. 10. The process of claim 1, wherein this process is conducted at a temperature ranging from 0 to 50 ° C.
11. 11. The process of claim 1, wherein step (a) is conducted at a pH greater than 9.
12. 12. The process of claim 1, wherein step (b) is conducted at a pH greater than 8.4.
13. 13. The process of claim 1, wherein the concentration of the somatotropin, in step (a), ranges from 1 to 10 grams / liter.
14. 14. The process of claim 1, wherein the reducing agent is added to the aqueous solution, in step (a).
15. 15. The process of claim 14, wherein the reducing agent is 2-mercaptoethanol, cysteamine, glutathione, cysteine or a redox mixture thereof.
16. 16. The process of claim 14, wherein said reducing agent is 2-mercaptoethanol, used in a concentration ranging from 0.001% to 0.5%.
17. 17. The process of claim 1, wherein a reducing agent is added in step (b).
18. 18. The process of claim 17, wherein the reducing agent is 2-mercaptoethanol, cysteamine, glutathione, cysteine or a mixture thereof.
19. 19. The process of claim 1, wherein the reducing agent is 2-mercaptoethanol, used at a concentration ranging from 0 to 0.25%.