The method of isolation of recombinant somatotropin

 

(57) Abstract:

Usage: biotechnology, biochemistry. The inventive recombinant somatotropin separated from the solution containing impurity proteins and other impurities, by directly adding an amine or Quaternary ammonium compounds to the solution in sufficient quantity for the selective deposition of impurity proteins with high molecular weight. The precipitate proteins with high molecular weight are removed, and then the solution is treated to remove proteins with low molecular weight and other deproteinised impurities. 5 C. p. F.-ly. table 2. Il.

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

The basis of the invention

Methods of producing recombinant proteins are well known in the art; the heterologous DNA segments that encode this protein are introduced into the microorganism host using recombinant DNA technology. By growing transformati microorganisms under conditions which induce expressioni, interferons, somatomedin and the like.

Unfortunately, heterologous proteins obtained using transformant microorganisms are often not biologically active, as they do not form needlecase tertiary structure during transcription inside the nucleus. Geterologichnaya proteins have a tendency to the formation of aggregate that are recognized within the cell as "body turn". These bodies include can be caused by the formation of covalent intermolecular disulfide bonds that connect together several protein molecules with the formation of insoluble complexes. Body inclusions typically contain a large part of heterologous proteins and a small fraction of contaminating proteins of the microorganism host.

Have developed several processes for the extraction of inclusion bodies of microorganisms and transformation of heterologous proteins contained in them, in proteins with native bioactivity consistent with natural source or non-recombinant proteins. These processes usually involve the destruction of the cells of the microorganism, Department of cell inclusions from the cell residue, dissolving the protein bodies including the l enable to remove insoluble contaminants, the removal of the denaturing agent/detergent that allows heterologous proteins to fold into a bioactive tertiary conformation, and separating the protein from contaminating proteins, which remain in solution.

Experts know several schemes purification of recombinant protein, following this General method: U.S. patent N 4511503 and N 4518526-Olsen et al and U.S. patent N 4511502 and N 4620948 Builder et al discloses a multi-stage methods, in which (I) the body include open multi-methods, in which (I) inclusion body dissolved in a strong denaturing agent (2), insoluble contaminants are removed from the solution solubilizing proteins, (3) a strong denaturing agent is replaced by a weak denaturing agent, (4) protein gets the opportunity to fold by oxidation of sulfhydryl groups to disulfide bonds using molecular oxygen and a catalyst, usually metal cations or tetrathionate sodium, and (5) the protein is separated from other contaminating proteins using techniques of membrane separation or chromatographic methods.

Raush et al, U.S. patent N 4,677,196, incorporated herein by reference, discloses a special method of cleaning and activation FTUS bodies in SDS, remove excess SDS from the solution using dialysis or other suitable method, chromatography was carried out SDS-protein solution on ion-exchange resin for separation of protein.

All these known methods have a common problem. The obtained protein solution, when it is possible detergent/denaturing agent, contains recombinant protein contaminating proteins with low molecular weight, deproteinize pollutants, and contaminants proteins with high molecular weight; contaminating proteins with high molecular weight for the most part are dimers, oligomers and aggregates of recombinant protein, but also include non-recombinant proteins of cellular life. Often it is difficult, requires time-consuming and expensive to separate the recombinant protein from these pollutants, particularly recombinant protein dimers, oligomers and aggregates. Methods of chromatographic and membrane separation can be effective for the separation of recombinant proteins, but are cumbersome, lengthy, expensive and often give low interest output selection protein.

New and improved ways for easy, fast and inexpensive selection of recombi the major weight are therefore necessary.

Fig. 1-4 are a graphical representation showing the effect of concentration on the allocation of porcine somatotropin and the ratio of polymer/monomer (P/M) determined by gel permeation chromatography (GPC).

Thus, the subject of the present invention is the provision of a new method for easy, quick and cost allocation high yield of recombinant protein from protein solutions containing contaminating proteins with high molecular weight.

Another object of the present invention is to provide for the disposal of high molecular weight contaminating proteins from a solution of recombinant protein, thereby allowing easy, quick and inexpensive to isolate the recombinant protein.

These and other objectives are achieved by direct addition of amine or Quaternary ammonium compounds to solutions containing high molecular weight contaminating proteins and recombinant protein in quantities sufficient for the selective deposition of high molecular weight contaminating proteins. Preferred compounds cause precipitation of proteins having a molecular weight greater approximately one and a half times, che is tov, having a molecular weight greater about 1.5 times the molecular weight of the recombinant protein. Precipitation is separated from the solution, and remain recombinant protein, low molecular weight contaminating proteins and other deproteinize contaminants in solution. Recombinant protein is released from the solution using known techniques and is processed to obtain the desired protein product.

In the preferred embodiment the amine or Quaternary ammonium compound is added directly to the solution in amounts sufficient to obtain a solution with a concentration of 0.01 to 2 volume percent. Contaminating high molecular weight proteins are precipitated and removed from the solution by conventional methods such as filtration, centrifugation and the like. The resulting protein solution containing recombinant protein contaminating proteins with low molecular weight and other deproteinize pollutants further processed using conventional techniques such as chromatography for isolation of recombinant protein.

Other objectives, advantages and new features of the present invention will become apparent from the following detain, you wish to highlight in a relatively pure form and include proteins having the amino acid sequence of native proteins and their analogs and muteins", having substituted, deleted, moved, or otherwise modified sequences.

The term "recombinant somatotropin" (rST), used here, includes recombinant proteins having the amino acid sequence of the native hormone, amino acid sequence essentially the same to him or them abbreviations kind of sequence, and their analogs and muteins", having substituted, deleted, moved, or otherwise modified sequences. In particular, rST used here, includes a protein of the same order as pST, but having amino acids deleted from its aminoterminal end. Examples of such proteins include, but are not limited to, Delta-7 recombinant porcine somatotropin, Delta-4 recombinant bovine somatotropin and the like.

The term "contaminating protein with high molecular weight" or "protein contaminant with high molecular weight" used herein refers to proteins having a molecular weight, rotini low molecular weight" or "protein contaminants with low molecular weight", used here, refers to proteins having a molecular weight less than about 1.5 times the molecular weight of the recombinant protein.

The term "deproteinize pollutants", as used here, refers here to a relatively low molecular weight substances, such as precipitating agents, solubilizing agents, oxidizing agents, reducing agents, and the like, which are usually found in the protein solution.

According to the present invention provides a method for isolation of recombinant protein from a protein solution containing a protein contaminants with high molecular weight. The method includes adding an amine or Quaternary ammonium compounds to the solution containing contaminating proteins with high molecular weight and recombinant protein in quantities sufficient for the selective deposition of high molecular weight protein contaminants. Amines or Quaternary ammonium compounds preferably cause precipitation of proteins having a molecular weight more than about 1.5 times the molecular weight of the recombinant protein, particularly dimers recombinant protein oligomers and AGR provides an improved method of the lung, quick and inexpensive selection with high yields of recombinant proteins from a solution containing a protein contaminants with high molecular weight. In the preferred embodiment, the method provides for the selection of recombinant somatotropin (molecular weight about 20,000) by directly adding an amine or Quaternary ammonium compounds to solutions containing contaminating proteins with high molecular weight and recombinant somatotropin in quantities sufficient for the selective deposition of protein contaminants with high molecular weight. Amine or Quaternary ammonium compounds cause precipitation of proteins having a molecular weight of more than about 1.5 times the molecular weight of recombinant somatotropin (molecular weight greater than about 30,000), especially dimers recombinant somatotropin, oligomers and aggregates having a molecular weight greater about 2 times than the molecular weight of recombinant somatotropin (molecular weight of about 40,000 and above). The presented method therefore provides a method for the separation of recombinant somatotropin from his bioreactive dimers, oligomers and aggregates.

Dissolve granitelli with high molecular weight, and protein contaminants with low molecular weight, are useful in the present invention, are obtained using techniques known in the art. Usually protein inclusion body, which were produced by recombinant microorganisms, are treated to remove lipids, and cellular debris and the resulting relatively clean body inclusions, containing the recombinant protein and contaminating proteins, especially recombinant protein dimers with high molecular weight oligomers and aggregates, solubilizers in a strong denaturing agent or a detergent, such as guanidine hydrochloride, sodium dodecyl sulphate (SDS), Triton, and the like.

The resulting protein solution is separated from any insoluble materials and a strong denaturing agent or detergent is removed to obtain a protein solution containing recombinant protein with native bioactive configuration protein contaminants with high molecular weight, intramolecularly contaminating proteins and other deproteinize pollutants. Such solutions typically contain from about 1 to 50 mg/ml protein and from about 0.05 to 4 mg/ml recombinant prsume the invention for deposition of contaminated proteins with high molecular weight.

High molecular weight contaminating proteins are precipitated by adding an amine or Quaternary ammonium compounds, are removed from the solution of well-known methods such as filtration, centrifugation and the like. The resulting protein solution containing recombinant protein contaminating proteins with low molecular weight, and other deproteinize pollutants, if any, are further processed, as necessary, to remove low molecular weight contaminating proteins and other deproteinised pollutants such as precipitating agents, solubilizing agents, oxidizing agents, reducing agents, and the like. Typically, such deproteinize pollutants are removed by dialysis, chromatography or other appropriate methods, whereas low molecular weight contaminating proteins are separated from the protein by ion exchange or other types of chromatography.

A protein solution is further processed to obtain the protein or protein compositions suitable for its intended use, usually by lyophilization. These methods are well known to specialists.

Amine or Quaternary ammonium compound, a field which SUB>4)+NX-< / BR>
in which R1, R2, R3and R4that may be the same or different, are selected from the group consisting of linear or branched C8-C20-alkyl, linear or branched C8-C20substituted alkyl and hydrogen, provided that at least one among R1, R2, R3and R4is not hydrogen; and X is an anion, such as chloride, bromide, iodide, sulfate, sulfonate, nitrate, acetate and the like or

< / BR>
where R1is selected from the group consisting of linear or branched C8-C20of alkyl; R2-R6selected from the group consisting of linear or branched C8-C20-alkyl, linear or branched C8-C20substituted alkyl and hydrogen; R' represents alkylenes or arene group; and X is an anion, such as chloride, bromide, idiot, sulfate, sulfonate, nitrate, acetate and the like; or

< / BR>
where R1, R2, R3and R4that may be the same or different, are selected from the group consisting of linear or branched C8-C20of alkyl, linear or branched C3and R4is not hydrogen; R' represents alkylenes or arene group, and X represents an anion such as chloride, bromide, iodide, sulfate, sulfonate, nitrate, acetate and the like; or

< / BR>
where R1, R2, R3, R4and R5that may be the same or different, are selected from the group consisting of linear or branched C8-C20of alkyl, linear or branched substituted alkyl or hydrogen, provided that at least one among R1, R2, R3and R4is not hydrogen; R' represents alkylenes or arene group and X is an anion, such as chloride, bromide, iodide, sulfate, sulfonate, nitrate, acetate and the like; or

< / BR>
where is a group derived from Tallow R2, R3, R4and R5that may be the same or different, are selected from the group consisting of linear or branched C8-C20substituted alkyl or hydrogen; R' is alkalinous or Arroway group; and X is anion such as chloride, bromide, iodide, sulfate, sulfonate, nitrate, acetate, etc.

Preferred linking include dodecylamine hydrochlo ethyldiamine chloride and DUOMAC-T (N-tallow - 1,3-diaminopropan diacetate). The most preferred compounds include DUOMAC-T (N-tallow 1,3-diaminopropan diacetate) and trimethyloctadecylammonium chloride.

Although the number of amine or Quaternary ammonium compound required to cause precipitation, varies depending on the concentration of protein, protein characteristics, added compounds and the like; amine or Quaternary ammonium compounds are generally added to the solution in sufficient quantity to obtain the concentration of the solution from about 0.01 to volume percent of a compound, preferably of the solution from about 0,01 to 0.5 volume percent.

Recombinant proteins to be identified when using this way of their possession, may be any protein having a molecular weight of more than about 5000, which are produced by recombinant microorganisms usually in inclusion bodies. These proteins include somatotropin, insulin, somatomedin, somatostatin, prolactin, placental lactogen etc.

Most preferably the recombinant somatotropin (molecular weight about 20,000) are allocated using the method of the present invention. Recombinant ICI, bird, sheep or human recombinant somatotropin, most preferably porcine or bovine recombinant somatotropin.

Methods of production of these recombinant proteins are well known in the art, for example: U.S. patent N 4604359 and N 4332717 describe methods for obtaining recombinant human somatotropin; U.S. patent N 4431739 discloses a method of obtaining recombinant somatotropins; publication of the application for the European patent N 104920 discloses a method of production of recombinant porcine somatotropin; U.S. patent N 4443359 describes a method of podocytopenia recombinant bovine somatotropin; Schoener, Biotechnology, 3/2/: 151-54, describes a method of obtaining recombinant somatotropin, and Buell, Nucleic Asid Res. 13,1923 38 (1985) discloses a method of obtaining recombinant somatotropin C.

Also, the publication of a European patent application N 0103395 describes the design transformatora E. coli strain containing the first plasma, which encodes a Delta-9 (Ser) bovine somatotropin (growth hormone without his 9 N-terminal amino acids and having an additional serine residue at the N-end) under the control of the lambda PL promoter-operator and which has a Shine-Dalgamo region derived from bacteriophage mu. Transferre repressing protein. Repressing protein can be inactivated by raising the temperature to about 42oC inducyruya thereby the expression of Delta 9 (Ser) bovine somatotropin. Transformer strain of this type, E. coli HB101 (PL-mu-Delta 9 (Ser) bovine somatotropin and RS) was deposited with American Type Culture Collection (ATSS), Rocxuille, MD and defined N 53030.

Design similar to the transformer strain that encodes the production of Delta 7 porcine somatotropin (porcine somatotropin without his first 7N-terminal amino acids) is described in the publication of a European patent application N 0104920. Transformer strain of this type, E. coli HB101 (Pl-mu-Delta 7 pork somatotropin and RS) deposited ATTSS and assigned the number N 53031.

Strains 53030 and 53031 are profilic" producers Delta 9 (Ser) bovine somatotropin and Delta 7 porcine somatotropin, respectively. In both cases, the expressed protein is isolated in cells in the form of insoluble inactive inclusion bodies, which are visible under a microscope. Other ways for many similar proteins known to experts.

In the preferred embodiment a solution of recombinant somatotropin, containing about 1 to 50 mg/ml total protein and approximately 0.05 to 2 mg/ml of PE is high molecular weight. The precipitate is removed by centrifugation and the recombinant somatotropin is released from the resulting solution, and uses a conventional means, as described above.

Although the above-described allocation method is aimed at the selection of recombinant protein, the method is put to the Department and the allocation of precombination proteins. For example, a solution containing a mixture of (1) and "useful or necessary" protein", (2) high molecular weight proteins (molecular weight of more than about 1.5 times the molecular weight of the useful protein) and (3) proteins with a low molecular weight (molecular weight less than about 1.5 times the molecular weight of the useful protein is processed according to the present invention for the deposition of proteins with high molecular weight and thus the separation of proteins with high molecular weight proteins with a low molecular weight and healthy protein. Proteins with high molecular weight are separated from the solution and discarded or optionally processed further; proteins with high molecular weight can be separated from the sediment by re-dissolving the precipitate and selection of proteins from solution.

Useful protein is separated on the La obtain a protein product. Proteins with low molecular weight, which are separated from the useful protein or discarded or further processed as desired. Usually proteins with low molecular weight can be separated from the useful proteins using chromatography or other means, suitable for separation of proteins with similar molecular weight. Specialists are well aware many ways the separation of these proteins, which are equally applicable in the present invention.

The invention is described in General, the following examples are given as particular embodiments of the invention and to demonstrate the practical application and benefits. It is clear that the examples are given for illustration and are not intended to limit the specification or the claims. In particular, inclusion body used in the experiments were obtained from transformed E. coli strains that produce Delta-7 porcine somatotropin. Body inclusions were isolated from the E. Coli host strain HB101 transformed first plasmid (Pl-mu-Delta-7 S) that encodes a Delta-7 the rST and the second plasmid, (RS) that encodes a temperature-sensitive protein repression lambda phage. Many other strains of microorganisms produce obretenii. Similarly, the methods of cultivation of these microorganisms for the production of inclusion bodies.

Example 1. Recombinant porcine somatotropin (gr.ST) was isolated from inclusion bodies of the microorganism by (1) dissolving the inclusion bodies in sodium dodecyl sulfate (SDS), (2) allocation of insoluble contaminants from a solution, and (3) allocation of SDS solution to allow gr.ST take the bioactive configuration. The resulting protein solution contained gr. ST, high molecular weight protein contaminants; low molecular weight contaminating proteins and other deproteinize pollutants. This solution was subjected to membrane separation for cleaning. Membrane separation has removed some of the high-molecular impurities, but a significant amount of high molecular weight impurities remained; was further purification.

20 milliliters (ml) of sample protein solution containing a protein contaminants gr.ST high molecular weight contaminating proteins with low molecular weight and other deproteinize pollutants were added to 50 ml beakers, which were placed in the ice filled container to maintain the temperature between 5 to 1 is a, and commercially available compounds amines a-1 and AA) was added to the sample using a 1 ml or 3 ml syringe. Reagents in each beaker was gently mixed using a coating Teflon stirrer for about 1 to 3 minutes. Sediments were formed almost immediately; the slower the speed of adding (dropwise) precipitator gave a purer product (the lower the ratio P/M). Similarly, a higher temperature (25oC) gave higher allocation. (However, in reality, in practice, operation at higher temperatures can be feasible because of the problems associated with microbial pattern). The contents of each beaker was centrifuged and supernant was filtered through an 0.2 micron filter to obtain a sample for gel chromatography (BPC).

Left overnight centrifuged tolerant didn't show any deposition, indicating that the reaction proceeded completely. The supernatant was analyzed using gel-penetrating chromatography and the data were then used to calculate the pST-selection and relationship polymer/monomer (P/M) in superatom product.

The results show that was allocated when the data connection of the P-14V and 52-267 were also tested, but they refused and preferred dodecylamine hydrochloride and a-1 or due to a lower allocation, the higher the ratio P/M, or because the required dosage. The test compounds are presented in table 1.

Example 2. Selected amine compounds a-1 and dodecylamine hydrochloride example 1 and a few others, including trimethyl dodecyl -, hexadecyl and octadecyl ameriglide, Duo-mac-T(N-tallow-1,3 - diaminopropane diacetate), aR-QUaD-2c-75(dimethyldecanamide chloride) and hexylamine were tested at various concentrations for a few "pilot"plant samples that were not subjected to membrane treatment. Except hexylamine, each of the above reagents resulted in precipitation of the protein. However differed selectivity and allocation pST. Among dodecyl -, hexadecyl-and octadecylammonium chloride, the concentration of reagent required for the selective deposition pST-polymer was a function of the length of the carbon chain in which at 18 carbon atoms octadecylphenol connection required only 0.02% (by volume solution), while 16 carbon atoms required to 0.03% and about 0,04-0,06% to 12 carbon atoms dolezelova connection. If the same ratio of P/M in the product, pST widely, DIMAS-T concentration ranges between 0,08-0,12% proved to be the most promising indicator of selectivity and allocation.

Table 2 represents the sum of the data for testing the deposition obtained using DIMAS-T and TDS with different groups of recombinant protein. Selection (75-82%) is more efficient than the efficiency of allocations when using methods of membrane cleaning. In addition, the precipitation process of the present invention is much easier and free from difficulties. Because the products are usually very valuable, small improvements in the efficiency of allocation yield greater economic benefits. The effect of the concentration of DIMAS-T pST-selection and the ratio P/M was studied in detail for several other types of crude recombinant protein. Show some typical results are presented in figure 1, there is a link between pST-allocation and ratio R/M, then there is a more efficient allocation associated with a higher ratio P/M, whereas the desired ratio of P/M in the pre-purified product is about 0.5 or less, the concentration of DIMAS-T must be chosen to satisfy this condition with the most high is Nasirova using analysis of tissue binding, isoelectric focusing (IEF) and liquid chromatography high pressure (HPLC). Analysis of tissue binding showed that 98% pST in relation to the standard sample pST was active, and suggested that pST was not are denatured. Similar tests IEF and HPLC showed no difference between treated DIMAS-T and unprocessed samples, indicating unchanged the structure of the pST.

Obviously many possible modifications and variations of the present invention in light of the procedures described above. Therefore, it should be understood that within the scope of the attached claims, the invention can be used in practice differently than described in this specification.

1. The method of isolation of recombinant somatotropin from a solution containing somatotropin, impurity proteins and other impurities, including extraction from transformed cells Taurus inclusion, their dissolution with formation of a solution containing active recombinant somatotropin, and the specified cleaning solution from impurities, characterized in that the purification of the target product from the impurity proteins with molecular weight of not less than 1.5 times the mass of the recombinant somatotropin, ooyama of dodecylamine hydrochloride, trimethyloctadecylammonium chloride, trimethyloctadecylammonium chloride, trimethylphenylammonium chloride, trimethylammonium chloride, dimethylammonium chloride, Dimas-T (N-tallow-1,3-diaminopropane) and ARQUAD-2C-75 (dimethyldecanamide chloride) to a solution in a quantity sufficient for the selective deposition of impurity proteins with high molecular weight.

2. The method according to p. 1, characterized in that the amine or compound Quaternary ammonium compounds are selected from the group consisting of Dimas-T(N-tallow-1,3-diaminopentane) and trimethyloctadecylammonium chloride.

3. The method according to p. 1, characterized in that the concentration of total protein in the solution is about 1 to 50 mg/ml, and the concentration of recombinant growth hormone in solution is 0.05 to 4 mg/ml

4. The method according to p. 1, characterized in that the amine or the connection of the Quaternary ammonium type in the quantity necessary to obtain a final concentration of 0.02 to 0.15%

5. The method according to p. 1, characterized in that the recombinant somatotropin is a growth hormone bull, pigs, birds, sheep or human.

6. The method according to p. 1, characterized in that the recombinant somatotropin is a growth hormone pig

 

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