The method of selection of the recombinant polypeptide with the sequence of somatotropin

 

(57) Abstract:

Usage: biotechnology, genetic engineering. The inventive obtained using recombinant DNA technology somatotropin extracted from transformed cells, dissolve and reactivit using known techniques to the solution re-activated protein at a pH of 9.0 and 9.8 add salt or calcium, or magnesium, or barium, or strontium in an amount necessary to obtain a final concentration of from 0.4 to 2.5%, incubated the reaction mixture under stirring until complete precipitation of the high molecular weight protein impurities, the residue is discarded, and the resulting solution is subjected to further purification. 4 C.p. f-crystals, 2 tab.

The invention generally relates to methods for isolating recombinant proteins, in particular to a method for isolation of recombinant proteins from protein solutions containing high molecular weight contaminating proteins.

Methods for obtaining recombinant proteins are well known, so the heterologous DNA segments encoding a specific protein, using techniques of biotechnology introduce microorganisms-hosts. The cultivation of microorganisms of the transformants under conditions that induce the expression of proteins by the P CLASS="ptx2">

Unfortunately, heterologous proteins produced by microorganisms-transformed, often biologically inactive, because these proteins do not coincide with the corresponding tertiary structure in their transcription in the nucleus. Heterologous proteins tend to form aggregates that can be recognized in the cells as "body turn". Such inclusion body may also result from the formation of covalent intermolecular disulfide bonds, which Niccolo protein molecules are associated with each other with formation of insoluble complexes. Body inclusions typically contain mostly heterologous proteins and a small fraction of contaminating proteins of the microorganism host.

Created several methods of extraction from microorganisms tel inclusion and transformation contained heterologous proteins in proteins with native biological activity comparable with the activity of related natural or non-recombinant proteins. Such methods usually involve the destruction of cells of the microorganism, the separation of inclusion bodies from cell debris, protein solubilization of inclusion bodies in denaturant-detergent releasing protein, the Department of heterologous proteins tel whichnis bioactive protein tertiary conformation and separation of protein from remaining in the solution contaminating proteins.

There are several schemes purification of recombinant protein using the above General procedure: U.S. patents NN 4511503 and 4518526 (01son, and others) and U.S. patents NN 4511502 and 4620948 (BuiIoer and others) reveal multistage ways in which (1) the body include solubilizers in strong denaturant and the reducing agent, (2) insoluble impurities are removed from the solution solubilizing protein, (3) strong denaturant replace weak denaturant, (4) perform protein straightening by oxidation of sulfhydryl groups to disulfide bonds using molecular oxygen and a catalyst, usually of metal cations and tetrathionate sodium, and (5) separating the protein from other contaminating proteins using the methods of separation on the membrane or chromatographic methods.

In U.S. patent N 4677196 (Rausch and others), given here as a reference, discloses a specific method of cleaning and activation of protein, which is a variation of the above General scheme. The method involves the solubilization of the inclusion bodies in a VAT, removal of excess VAT from solution using dialysis or other reasonable procedures, chromatography was carried out of the protein solution in the VAT for inhibiting ions of the resin and the chromatography was carried out resulting solution on anyoneeven is, obtained after removal denaturant detergent contains a recombinant protein, a low molecular weight contaminating proteins, non-protein impurities and high molecular weight protein impurities, while high molecular weight protein impurities often consists mainly of dimers, oligomers and aggregates of recombinant protein, but also include non-recombinant proteins from hydrolysis of the cell. Often the separation of the recombinant protein from these impurities, in particular dimers, oligomers and aggregates of recombinant protein is achieved with difficulty, time consuming and expensive. Methods of chromatography and separation membranes can be effective in the separation of protein from impurities, but such methods are cumbersome, time-consuming, expensive and often lead to low interest yield allotment of protein.

Thus, there is a need for new and improved methods are simple, rapid and inexpensive selection with high yields of recombinant protein from solutions containing high-molecular protein impurities.

Thus, the objective of the present invention is to provide a new method is simple, rapid and inexpensive selection with high yields of recombinant proteins from Belk is Oia is to provide a method for removal of high molecular weight impurity proteins from a solution of recombinant protein, that allows easy, fast and inexpensive to isolate the recombinant protein.

These and other objectives of the invention achieves the direct addition of metal salts IIA Group to the solution containing high molecular weight impurity proteins and recombinant protein in a quantity sufficient for the selective deposition of high molecular weight protein contaminants. Salts induce the preferential precipitation of proteins with a molecular mass of approximately one and a half times greater than the molecular weight of the recombinant protein, in particular dimers, oligomers and aggregates of recombinant protein. The precipitate is separated from the solution, leaving the solution of the recombinant protein, low-molecular-weight impurity proteins and other non-protein impurities. Recombinant protein isolated from the solution by the known methods and processing gain of the target protein product.

In a featured embodiment of the invention IIA metal salt Groups are added directly to the solution in sufficient quantity to create a 0.1 to 10% (about) of the solution. High molecular weight impurity proteins are precipitated and removed from the solution by conventional means, such as filtration, centrifugation, etc. of the Obtained protein solution, containing what she processed using normal procedures, for example chromatography with separation of the recombinant protein.

Other objectives, advantages and new features of the present invention will become apparent from the following detailed description of the invention.

The term "recombinant protein" as used here, the value of the indicated protein, obtained by recombinant methods, which I want to highlight in a relatively pure form, including proteins with the amino acid sequence of native proteins and their analogues and Malinov characterized substituted, subject to the deletion, replacement or otherwise modified sequence.

The term "recombinant somatotropin" (PCT) used here includes recombinant proteins with the amino acid sequence of native somatotropin, aminosilanes sequence essentially similar sequence of native somatotropin or shorter sequence, as well as their analogues and mutiny characterized substituted, subject to deletions, replaced or otherwise modified sequence. In particular, the PCT used here, the value includes a protein with the same sequence as the natively shall include, but without limitations only by them: recombinant Delta-7 - somatotropin pigs, recombinant Delta-4-somatotropin cows, etc.

The term "high molecular weight impurity proteins or macromolecular protein impurities" as used here, the value applies to proteins with molecular mass about 1.5 times the molecular weight of the recombinant protein, in particular dimers, oligomers and aggregates of recombinant protein, whose molecular weight is about twice the molecular weight of the recombinant protein.

The term "low-molecular-weight impurity proteins or low-molecular-weight protein impurities" as used here, the value of the indicated proteins with a molecular mass of approximately 1.5 times lower than the molecular weight of the recombinant protein.

The term "non-protein impurities" as used here, the value applies to a relatively low molecular weight substances, such as precipitators, solubilizing means, oxidizing agents, reducing agents, etc., are usually present in protein solutions.

According to the present invention provides a method of isolation of recombinant protein from a protein solution containing high molecular weight impurity proteins. The method concludes the s proteins and recombinant protein in quantities sufficient for the selective deposition of high molecular weight protein contaminants. Metal salt IIA Group mainly induce the precipitation of proteins, whose molecular weight is about 1.5 times the molecular weight of the recombinant protein, in particular dimers, oligomers and aggregates of recombinant protein, the molecular weight of which is approximately 2 times higher than the molecular weight of the recombinant protein. The method is a superior method is easy, quick and cost allocation with high yields of recombinant proteins from solutions containing high-molecular protein impurities.

In a featured embodiment of the invention provides a method of separation of recombinant somatotropin (molecular weight about 20,000) direct addition of metal salts IIA Group to solutions containing high molecular weight impurity proteins and recombinant somatotropin in quantities sufficient for the selective deposition of high molecular weight protein contaminants. Metal salt IIA Group mainly induce the precipitation of proteins with a molecular mass of about 1.5 times the molecular weight of the recombinant somatotropin (with molecular weight more than about the 40000 and above). Thus, the present method is a method of separation of recombinant somatotropin from its biologically inactive dimers, oligomers and aggregates.

Solutions containing recombinant protein, non-protein impurities, high molecular weight protein impurities and low molecular weight protein impurities and applicable in the present invention, receive well-known methods. As a rule, the protein inclusion body produced by recombinant microorganisms, is treated to remove lipids and cellular debris, and the resulting relatively clean body inclusions, containing the recombinant protein and impurity proteins, in particular high molecular weight dimers, oligomers and aggregates of recombinant protein, solubilizers in strong denaturant or detergent, such as guanidine hydrochloride, nitrilotriacetate (VAT), Triton, etc.

The obtained protein solution is separated from any insoluble substances and after removal of the strong denaturant or detergent get the protein solution containing the recombinant protein translated in their native bioactive configuration, high molecular weight protein impurities, low-molecular-weight impurity proteins and other non-protein PR> The ratio of polymer to monomer (S/MB)in this solution includes a ratio by weight of aggregates, including the empty peak to the monomer, which is a measure of the purity of the solution. The solution typically encompasses "pretreatment", where the ratio P/M is reduced to approximately 0.5 or below. Then the solution may be subjected to further cleaning and processed into the final product, as a rule, the use of EAE-Sepharose or other acceptable speakers. The present invention provides a method of removing high molecular weight impurity proteins with simultaneous achievement of a higher degree of selection of the monomer and lower ratios N/M

Metal salt IIA Groups added to this solution, in accordance with the present invention, with the purpose of the deposition of high molecular weight impurity proteins. High molecular weight impurity proteins precipitated by adding metal salts IIA Group, removed from the solution by conventional means such as filtration, centrifugation, etc. of the Obtained protein solution containing the recombinant protein, low-molecular-weight impurity proteins and other non-protein impurities, if any, is subjected to further processing as necessary with the removal of low-molecular protanomaly etc. Typically, these non-protein impurities are removed by dialysis, chromatography or other appropriate methods, while low-molecular-weight impurity proteins are separated from the protein by ion exchange chromatography or its variant.

The protein solution is subjected to further processing to obtain the protein or protein compositions, suitable for the intended use, usually by lyophilization. Such methods are well known.

Salts of metals of Group IIA, applicable in the present invention includes all salts of elements of Group IIA of the Periodic table: as beryllium, magnesium, calcium, strontium, barium and radium. Featured compounds include salts of magnesium, calcium, barium and strontium.

The most recommended connection of the present invention include sulfate (SO4), chloride (Cl2) and nitrate (NO3) magnesium, calcium, barium and strontium. Featured compounds include calcium sulfate (CaSO4), anhydrous CaSO4, hemihydrate CaSO4, calcium nitrate (Ca(NO3)2), calcium lactate, magnesium sulfate (MgSO4), magnesium chloride (MgCl2), barium chloride (BaCl2) and strontium chloride (SrCl2).

Although the number of metal salts IIA Group necessary for deposition, m IIA metal salt Groups are usually added to the solution in amounts sufficient to create a concentration of the compound in the solution is 0.1-10%, preferably 1-3%

Recombinant protein secreted by the method of the present invention may be any protein with a molecular weight of more than 5000, produced by recombinant microorganisms, usually in inclusion bodies. Including somatotropin, insulin, somatomedin, somatostatin, prolactin, placentalia lactogen etc.

Most preferably, if the method of the present invention produce recombinant somatotropin (molecular weight of about 20,000. Recombinant somatotropin can be recombinant somatotropin from any source, but preferably is a bovine, swine, poultry, sheep, human recombinant somatotropin, most preferably porcine or bovine recombinant somatotropin.

Methods of obtaining such recombinant proteins are well known, see for example, U.S. patent NN 4604359 and 433717, revealing the means of obtaining human recombinant somatotropins, U.S. patent NN 4431739, disclosing a method of obtaining a recombinant somatotropins, the application for the European patent 0104920, disclosing a method of obtaining a recombinant porcine somatotropin, patent Hide a method of obtaining a recombinant somatotropin, and work Buell, Nucleic Acid Res. 13, 1923-38 (1985), revealing a method of obtaining a recombinant somatomedin C.

In addition, the application for the European patent, publication 0103395, describes the construction of strain-transformant E. coti, containing the first plasmid encoding a bullish Delta-9-(Ser)-somatotropin (growth hormone, reduced to their 9 N-terminal amino acids and additional serine residue in the N-end) under control of the lambda PL promoter-operator and has an area Shine-Dalgarno originating from bacteriophage mu. The transformer also includes a second plasmid (RS 1857), coding for the production of temperature-sensitive protein-repressor RS 1857. Protein-repressor can be deactivated by raising the temperature to about 42oWith induction in the expression bullish Delta-9-(Ser)-somatotropin. Strain the transformant of this type /E. coti HBIOI (PL-mu-Delta-9-(Ser)-somatotropin cows and RS 1857) deposited in the American type culture collection (ATSS), Rockville, PCs CBM under registration N 53030.

The design is similar to the strain-transformant encoding the production of pork Delta-7-somatotropin (porcine somatotropin, reduced to their 7 N-terminal amino acids) described in C the pigs and RS 1857) deposited in ATSS under registration N 53031.

Strains 53030 and 53031 are productive producers respectively bullish Delta-9-(Ser)-somatotropin and pork Delta-7-somatropina. In both cases, the expressed protein is sequestered in the cell in the form of insoluble, biologically inactive, visible under the microscope of inclusion bodies. There are other ways of obtaining many other similar proteins.

In a featured embodiment of the invention a solution of recombinant somatotropin containing 1-50 mg/ml total protein and 0.05-2 mg/ml recombinant somatotropin, is treated with 1-3% CaSO4with the deposition of high molecular weight protein contaminants. The precipitate is removed by centrifugation and the recombinant somatotropin separated from the resulting solution using the above procedures.

Although the above method of selection is aimed at the selection of recombinant protein, the method is equally applicable for the separation and allocation of non-recombinant proteins. For example, a solution containing a mixture of (1) "useful or target protein" (2) high molecular weight proteins (molecular weight, about 1.5 times the molecular weight of the useful protein) and (3) low molecular weight proteins (molecular weight, about 1.5 times smaller than the separation of high molecular weight proteins from the valuable protein and low molecular weight proteins. High molecular weight proteins separated from the solution and discarded or, if desired, subjected to further processing, high molecular weight proteins can be isolated from sediment re-dissolution and extraction of proteins from solution.

Useful protein is separated from low molecular weight proteins by conventional means and, if desired, subjected to further processing to obtain the protein product. Low molecular weight proteins, which are separated from the useful protein, discarded or, if desired, subjected to further processing. Generally, low molecular weight proteins can be separated from the useful protein chromatography or otherwise, applicable for the separation of proteins with similar molecular masses. Many of these methods of separation of proteins is well known experienced and equally applicable in the present invention.

After the invention disclosed in General terms, the following examples are given as particular embodiments of the invention and to show its practical features and its benefits. It is clear that the examples are given only for illustrative purposes and in no way are intended to limit the description or subsequent claims. In particular, prototropic. Body inclusions isolated from E. coti strain-host HBIOI transformed first plasmid (PL-mu-Delta-7-TMS) that encodes a Delta-7 cSt, and the second plasmid (RS 1857) that encodes a temperature-sensitive protein-repressor of lambda phage. Many other microorganisms produce inclusion body containing recombinant proteins of many types that will work in this invention. Similarly, the methods of cultivation of such microorganisms to obtain inclusion bodies is well known.

Example 1. Recombinant porcine somatotropin (rst) was isolated from inclusion bodies (1) solvent tel inclusion in nitrilotriacetate (VAT) in carbonate buffer (25 mm NaHCO3and 21 mm Na2CO3), (2) removing from the solution of insoluble impurities, (3) adding an oxidant to oxidize rst and (4) removing from the solution the VAT in order to allow rst to take bioactive configuration. The obtained protein solution contains rst, high molecular weight impurity proteins and other non-protein impurities.

To the obtained solution of the protein with the restored configuration containing 0,67 mg/ml Monomeric rst, with P/M ratio of 5.1 (pH 9,8 at 22-25oC) add CaSO42H2O to a concentration of 2.5% (by weight of the e monomer rst and P/M ratio in the supernatant analyzed by liquid chromatography for rapid determination of proteins (GHBB) Superose-12. The results show that the ratio P/M decreased to 0.3, and the selection of the monomer is 83% of the Obtained data show that the protein aggregate impurities and high molecular weight impurity proteins selectively deposited CaSO42H2O separation in the solution of monomers.

Example 2. Reproduced in example 1, but with the use of other calcium salts: anhydrous CaSO4, hemihydrate CaSO4, calcium nitrate (Ca(NO3)2and calcium lactate. The results are shown in table.1.

The data table. 1 shows that the aggregate impurities and high molecular weight impurity proteins selectively deposited adding salts listed.

Example 3. Reproduced in example 1, but with the use of salts of alkaline earth metals: magnesium, barium and strontium. The results are shown in table.2.

The data table. 2 shows that the aggregate impurities and high molecular weight impurity proteins selectively deposited listed in table salts of alkaline earth metals.

Example 4. To the protein solution with the restored configuration of example 1, containing 0.4 mg/ml rst monomer with the N/M ratio of 4.7, add CaCl22H2O to a concentration of 0.4% (wt./about) and about 9% and after centrifugation for 10 minutes at 5000G collect the supernatant, which analyze GHBB on Superose-12.

In the P/M ratio was reduced to 0.4, and the selection of the monomer is of 92.1%, These data suggest that CaCl22H2O besieging protein aggregates of high molecular impurity proteins selectively, leaving in the monomer solution.

Example 5. The protein solution with the restored configuration example 1 concentrate about half and diafiltrate regarding 80 mm ethanolamine buffer at pH 9. To diafiltration protein solution containing 0,67 mg/ml Monomeric rst, with P/M 3.5 add CaCl22H2O to a concentration of 0.4% (wt./about) and stirred for 30 minutes at 22-25oC. by Centrifugation for 10 minutes at 5000G separate the supernatant, which is analyzed by GHBB on Superose-12.

As a result, the ratio P/M is reduced to 0.22, and the selection of the monomer is of 91.5% the data show that CaCl22H2O selectively besieging protein aggregates of high molecular impurity proteins in ethanolamine buffer system.

Obviously, in light of the above, there are numerous modifications and variations of the present invention. In this regard it must be emphasized that the presented examples.

1. The method of selection of the recombinant polypeptide with the sequence of somatotropin from a solution containing an impurity of high molecular weight proteins, involving the extraction of heterogeneous protein from transformed cells, its dissolution and reactivation, Department of macromolecular impurities and final purification of the target product, wherein after receiving the solution reactivated somatotropin to him at pH 9,0 9,8 add salt, or calcium, or magnesium, or barium, or strontium in an amount necessary to obtain a final concentration of 0.4 to 2.5% of the incubated mixture with stirring until complete precipitation of proteins with a molecular mass of at least twice the molecular weight of somatotropin, the precipitate is separated and the solution is subjected to further purification.

2. The method according to p. 1, characterized in that the concentration of total protein in the solution is 1 to 50 mg/ml, and the concentration of secreted polypeptide 0.05 to 2.0 mg/ml

3. The method according to p. 1, wherein the salt is selected from the group including sulfate, chloride and nitrate.

4. The method according to p. 3, characterized in that the salt is chosen from the group comprising anhydrous calcium sulfate, dehydrative magnesium, the chlorides of magnesium, barium and strontium.

5. The method according to p. 1, characterized in that the quality of the salt used calcium lactate.

 

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