Method of extracting somatotropic protein from light-refracting bodies of host cells

专利摘要:
A method for solubilization and naturation of somatotropin protein from refractile bodies of host cells is disclosed. The method embraces the discovery that an aqueous urea or dimethylsulfone solution can be effectively used to solubilize refractile bodies containing such somatotropin protein. Once solubilized, somatotropin protein can be natured in a urea or dimethylsulfone solution by contacting the solution with a mild oxidizing agent for a time sufficient to result in the formation of the disulfide bonds. Naturation can efficiently occur even at high protein concentration, in an impure preparation and in the absence of reducing agent.
公开号:SU1542402A3
申请号:SU4022043
申请日:1986-02-21
公开日:1990-02-07
发明作者:Эндрю Бэнтл Лерри；Уиллиэм Митчелл Джеймс；Брадлей Сторс Стифен；Тсуъеши Шимамото Грант
申请人:Монсанто Компани (Фирма)；
IPC主号:

专利说明:

This invention relates to medicine and can be used to isolate a somatotropic protein from E. Coli cells.
The purpose of the invention is to simplify the method and improve the quality of the target product.
Figures 1 and 2 show graphs of the total amount of solubilized protein versus urea concentration; on fig.Z - schedule of monomer yield depending on the concentration - / - x. urea
Part 1: Solubilization of bovine N-methionyl somatotropin (MBS), expressed in E. Coli.
Collected cells are torn, skipping them twice through a homogenizer
like Man ton-Gaul in homo genizer. The homogenate solution is subjected to low-speed centrifugation, with the result that light-refracting bodies containing MBS are precipitated. The supernatant is discarded, the light refracting bodies are re-weighed, washed and centrifuged again. The supernatant is again discarded and the light refracting bodies are prepared.
The light refracting bodies prepared in this manner at 25 ° C are exposed to different concentrations of an aqueous solution of urea at different pH values. All buffered solutions contain 100 mmol Tris base.
cm
Vani. The adjustment of the pH value is carried out by the addition of HC1. The final concentration of the light refracting bodies is 4 mg / ml of solution. The degree of dissolution is determined spectrophotometrically, taking the light refracting bodies completely proteinaceous and using an extinction coefficient of E 0.68 at 277 nm in a path length of 1 cm as a protein concentration indicator of 1 mg / ml. Outcome-; The total concentration is determined spectrophotometrically for a completely dissolved sample.
The results of the described experience are presented graphically (figure 1). These data confirm that adjusting the pH to provide alkaline conditions significantly increases the degree of solubilization.
Example 2. It is carried out analogously to Example 1, but the temperature is kept at 4 ° C and the pH value is regulated both towards the acid and towards the alkaline reaction. All buffered solutions contain 100 mmol-tris-bases. Adjusting the pH to acidic is accomplished by adding acetic acid.
The results of this experience are presented graphically (figure 2). Comparison of the data of figures 1 and 2 shows that at a constant concentration of the urea solution, and at a given pH value, the degree of solubilization at lower temperatures (4 ° C) is higher than at room temperature (25 ° C).
EXAMPLE (comparative). MBS-containing light refractory bodies are prepared according to Example 1. The light refracting bodies are mixed with 10 M urea solution without adjusting the pH value, so that the final body concentration is about 5.0 mg / ml. The solution is then mixed and, in order to establish equilibrium, it is kept at 4 ° C overnight. The degree of solubilization is then determined spectrophotometrically, which is only about 2.9 mg / ml. The initial concentration of the light refraction of the bodies is determined by adding a sufficient amount of a solution of urea in order to completely dissolve them and spectrophotometrically measure the solution completely.
five
0
50
five
0
5 -Q
five
renna solution. Then the initial concentration is reduced accordingly.
PRI me R B (comparative). MBS-containing light refractory bodies are prepared according to Example 1. Then mixed with an 8.0 M aqueous solution of urea without adjusting the pH so that the final concentration of the bodies is about 5.0 mg / ml. The solution is then mixed and, in order to establish equilibrium, it is maintained at 4 ° C overnight. The degree of solubilization is determined spectrophotometrically, which is about 2.4 mg / ml. The initial concentration of the light refracting bodies is determined by adding a sufficient amount of the urea solution to completely dissolve them and spectrophotometrically measuring the completely dissolved solution. Then the initial concentration is reduced accordingly.
Example 3. Light refracting bodies containing MBS and prepared as in Example 1 are mixed with unbuffered aqueous 4.5 M urea solution so that the concentration of light refractory bodies is about 66 mg / ml. The pH of the solution is increased from 7 to 11 with diluted sodium hydroxide solution. The clarified solution shows complete solubilization. The concentration of light refracting bodies determined in Example 1 by spectrophotometric analysis is 66 mg / ml.
Example 4. MBS containing light refracting bodies obtained in Example 1 were solubilized in an aqueous 7.5 M urea solution containing 100 mmol Tris at pH 10.5. The urea concentration is maintained at different levels, 100 mmol of tris is added, and the total protein concentration is maintained at about 1 mg / ml, as determined by spectrophotometric analysis of a completely dissolved sample, by adding a certain amount of the appropriate urea solution. Then, the dissolved MBS is oxidized, exposing the solution to air and stirring it throughout the day. The results are presented graphically (fig.Z). The monomer yield of MBS is indicated as weight percent of total MBS content. Optimum naturalness is achieved
jirpH urea concentration of about 4.5M (fig.Z).
Example 5. According to example 4, MBS containing light refracting bodies are solubilized in an aqueous 7.5 M solution of urea containing 100 mmol of Tris and with a pH value of 10.5. After solubilization, the solution is acidified with oxygen overnight with stirring. Data from BLC analysis show that the optimal yield of MPS monomer is achieved at a urea concentration of 3 M.
Example 8. Analogously to example 7, solubilize MPS at 4 ° C in an aqueous 7.5 M solution of urea with
100 mmol of tris is added up to a concentration of JQ, pH 11, adjusted by the addition of 4.5 M urea, followed by 90 mmol of tris. The preparation of the pH is adjusted to values within the range of solutions of three different concentrations from 10.5 to 8.5. Then, individual plants (20.40 and 80 mg of a (wet) pellet for thieves are oxidized with air oxygen with 1 ml of the indicated urea solution), stirring for 24 hours. Soder-15 Samples of solutions are diluted with 90 mmol monomer and oligomer is determined by tris and / or urea in order to obtain according to GC. Although the results are edicts--; neither is 3 M urea concentration and the fact that the dependence of the effective concentrations of MPS is 1 mg / ml. Diluted
MPS solutions with agitation are exposed to air at 4 ° C. for 56 hours. BHH analysis shows
The naturability from the pH value during the oxidation process is relatively even, but a tendency to increase in efficiency as the pH value is observed. In addition, the process catalyzed by the base oxide
the average yield of monomer MPS, equal to 69 wt.%.
Example 9. The similarity proceeds the faster, than with example 8, but the solubilization and
The alkaline reaction of the solution is more pronounced.
Example 6. To 850 ml of an aqueous 5.3 M solution of urea at 4 ° C are added 150 ml of the light refracting agent prepared according to Example 1. The pH of the resulting 4.5 M urea solution of the 50% by weight sodium hydroxide solution was adjusted to 11. The bodies completely dissolved, resulting in a total MBS concentration of about 12.4 mg / ml. The solution is then stirred at 4 ° C overnight for the purpose of MBS oxidation. In accordance with the data BLC-analysis of the oxidized solution in the result is the output of the monomer MBS, equal to 80 wt.%.
Example 7. Porcine S-methionyl somatotropin (MPS) is expressed in E. Coli. After isolating the MPS containing light refracting bodies by the described method, the latter are solubilized at 4 ° C in a 7.5 M solution of urea with a pH value of 11.0, adjusted by the addition of 90 mmol tris. Then a ball of light refracting material, weighing 1–13 mg (in the wet state) is dissolved in 1 m of the obtained urea solution. The samples are then diluted with 90 mmol of tris and / or urea in order to obtain a solution of urea with a concentration of 4.5; 3.0 and 2.0 M with a MPS concentration of 4 mg / ml, based on the weight of the wet bulb of the light refractory bodies. Then samples
the average yield of monomer MPS, equal to 69 wt.%.
Example 9. Conduct analog
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The reaction is carried out in the presence of 0.1 mmol 1,4-dithiothreitol. BLC-analysis shows the average yield of monomer MPS, equal to 66 wt.%.
Example 10. Three variants of BGH: A-la (, Ala, and Met, Val, 26, are expressed in E.Coli according to known methods. Some variants of BGH are solubilized and subjected to naturalization as in Example 1.
The light refracting bodies containing the corresponding BGH variants were isolated as in Example 1. Then, about 300 g (in the wet state) of bodies were weighed in water to obtain 1 liter of the suspension. The latter is added to 5 L of 9 M urea and 108 mmol of Tris, resulting in a solubilizing solution, including the corresponding BGH variant of 7.5 M of urea and 90 mmol of Tris at a pH of 10.5 and 4 ° C. After stirring for several minutes, the solubilization is completed. Then add 4 L slowly.
0 cold water to obtain a solution intended for naturalization, containing the corresponding BGH variant of 4.5 M urea and 54 mmol tris at a pH value of 10.5 and at 4 ° C. The solution is mixed and the corresponding BGH is oxidized, the solution is exposed to air for 48 hours. The solutions with oxidized BGH variants are subjected to BLC analysis, which
all three options BGH shows the output of the monomer 60-70 wt.%.
Example 11. The structural homology of the somatotropin protein obtained in the manner described, and the somatotropin produced by the pituitary gland, is determined by the method of Circular dichromistn. MBS and variant Ala., Somatotropin BGH are compared with bovine somatotropin. Samples are dissolved in a solution of 50 mmol sodium bicarbonate at a pH value of 9.5 and analyzed by the method described.
The data of this analysis show that the recombinant somatotropin obtained in the described manner after naturation has its own native conformation.
Spectral data are given in Table. 1 and 2.
Example 12. MBS containing refractory bodies were prepared as in Example 1 and mixed with unbuffered 1.0 M aqueous solution of urea at 4 ° C. The pH is adjusted to 12.1 by addition of sodium hydroxide and kept at this level. The clarification of the solution indicates the completion of the solubilization. The concentration of the light refracting bodies was determined analogously to example 1 using a spectrophotometric analysis method, which coc-v was applied at about 10 mg / ml.
Example 13. MBS containing refractory bodies were prepared as in Example 1. They were then dissolved in a 3.0 M dimethyl sulfone solution at 28 ° C (pH 11.8) with an MBS concentration of 2 mg / ml. As a result of air oxidation of dissolved MBS, a product is obtained which (judging by the degree of intermolecular bonding) differs only slightly from that obtained using a 4.5 M urea solution with a pH value of 11.3 (50 mmol tris) at 5 ° WITH.
The somatotropins solubilized and naturized in the manner described are then purified by standard chromatographic techniques. The bioactivity of somatotropins is shown by a positive reaction caused by them in tests based on rat growth (rat growth, bioassy).
In this case, the bioactivity of the heterologous samatotropin is determined by the relative

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five
five
a known amount of somatotropin material (for example, bovine or porcine pituitary somatotropin), establishes a relationship between the increase in weight of rats subjected to hypophysectomy, and different amounts of test material introduced into their bodies. The regression slope of the increase in body weight compared to the doses of the injected specific somatotropin material is compared to a known standard material (e.g., pituitary) and the relative bioactivity of the heterologous somatotropin material in U units / mg growth hormone is calculated.
Bovine N-methionyl somatotropin, combined and naturized according to the proposed method, is purified and then introduced into the organism of dairy cows. Dairy cows injected with such a drug produce 10–40 wt.% More milk than control animals.
The expression somatotropin includes mammalian somatotropins, such as human, ovine, porcine, and bovine somatotropins, and other somatotropins, such as avian, as well as systems including analogs and homologs of a naturally occurring protein that exhibits somatotropin-like bioactivity, such as prolactin and lactate.
Heterologous proteins are proteins that are not normally produced by host cells. Thanks to DNA recombination technology, it has become possible to express relatively large quantities of heterologous proteins from transformed host cells. However, these foreign proteins are often masked in insoluble light refractory bodies in the cytoplasm of the host cells.
Addition is the restoration of the total conformation of a protein, sufficient to effect oxidation properly. The addition process is carried out by reducing the denaturing effect of urea, if necessary, by lowering its concentration to the desired level to ensure the interaction of the amino acid sequence of the protein and to achieve a natural secondary and tertiary structure.
Oxidation is the formation of intramolecular disulfide bonds in order to obtain a stable native conformation that provides the desired biological activity.
A mild oxidizing agent is a substance that promotes the oxidation of sulfhydryl groups to form intramolecular disulfide bonds without oxidizing somatotropin, HGH is somatotropin
lazy other substituents of this protein. Although it is possible to use such mild agents as hydrogen peroxide, it is quite enough (and it is preferable) to use air for these purposes.
Biological activity is the ability of somatotropin to carry out its intended physiological response in vivo. In the absence of in vivo testing of a specific animal species, the biological activity can be determined by conducting suitable biological tests. A suitable bioassay for somatotropins of the proposed method is a bioassay for determining weight gain in rats. This test determines the bioactivity of somatotropin preparations in comparison with known preparations (for example, extracted native somatotropin), establishing a relationship between the degree of increase in weight in rats subjected to hypophysectomy, and different amounts of the preparation administered to the rat.
Optogenic somatotores are hormones secreted by the adenohypophysis (anterior pituitary gland) and stimulate skeletal growth and weight gain. Somatotropins usually contain 191 amino acid residues and have a molar mass of 2200.0 daltons. Full follower
the amino acid residues are established; solubilization is carried out in the presence of
Lena for somatotropins of various species, including humans and animals, for example birds (avian somatotropin), sheep
(sheep somatotropin), pigs (pork somatotropin), cattle (bovine somatotropin).
Table 3 shows the primary structure of different somatotropins visible. animals, with BGH - bovine somatotropin, PGH - pork somatotropin, OGH - sheep somatotropin, AGH - pti5
0
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human, the symbol X denotes a gap in the sequence and is inserted to illustrate the location of typical somatotropins. When numbering the amino acid residues of a specific sequence, this insertion is not taken into account, for example in BGH position 1 26- is Leu (or Val, as in the case of the allelic variant used in Example 10).
Tabulated relative amounts of specific amino acids based on the sequences described are listed in Table 4.

权利要求:
Claims (1)
[1]
Invention Formula
A method for isolating a somatotropic protein from the light-refracting bodies of host cells, including solubilization followed by naturalization and purification of the target product, characterized in that, in order to simplify the method and improve the quality of the target product by maintaining its native properties, the host cells Ina, E. coli bacteria are used, solubilization is carried out with 4.5-10 M solution of urea or dimethyl sulfone at pH 9-12.1 and -25 ° C, and naturalization is carried out in an aqueous solution of 2-5 M urea at pH 9 - -12.1 and a temperature of 4-25 ° C, where
or the absence of a reducing agent, natural, in the presence of a mild oxidizing agent.
I
max NM
A cm
278.2
15.042 ± 440
0.690
278.2
14.935 ± 216 0.680
Note. Em is the extinction coefficient at 278 nm.
A10 ° is the normalized absorption of an I mg / ml solution in a cell of size I, 0 cm at 278 nm.
291 285 265 221 about-helix
278.5
15,150 ± 100 0,693
table 2
13 154240214
Table 3
120
WONPhe-Pro-Ala-Mel-Ser-Leu-Ser-Gly-Leu-Phe-Ala-Asn-Ala-Va I-Leu-Arg-Al a-Gin-Hie-LeuPGH-ProSer ----
OCH
ACH-ProAsn
HCHThr-lle-ProArgAspMetHis-Arg
3040
BGHHis-Gln-Leu-Ala-Ala-Asp-Thr-Phe-Lys-Glu-Phe-Glu-Arg-Thr-Tyr-Ile-Pro-Clu-Gly-GlnPGH-TyrAla-
Ogh
AGHLeuGinTyr-Aip - HGHPheTyr-GlnGlu-AlaLys-Glu
5060
BGHArg-Tyr-Ser-X-Ile-Gln-Asn-Thr-Gln-Val-Ala-Phe-Cys-Phe-Ser-Glu-Thr-Ile-Pro-AlaPGHAlaAla
OGH-
AGHThrAsn-LysSerAlaTyr-
HGHLys --- Phe-Leu - ProThr-Ser-Leu-Ser - Thr7080
BGHPro-Thr-Gly-Lys-Asn-Glu-Ala-Gln-Cln-Lys-Ser-Asp-Leu-Glu-Leu-Leu-Arg-Ile-Ser-LeuPGHAsp-ArgValPhe
Ogh
AGHAsp-AspMet-GlyPhe
HGHSer-Asn-Arg-GluThrAsnGin
90,100BGHLeu-Leu-Ile-Gln-Ser-Trp-Leu-Gly-Pro-Leu-Gln-Phe-Leu-Ser-Arg-Val-Phe-Thr-Atn-Ser-
PGHVal
Oghr-
AGHVal ™ --ThrValTyrLysAsnHGH - GluVal --- Arg-Ser -: Ala
110120
BGHLeu-Val-Phe-Gly-Thr-Ser-Asp-Arg-X - Val-Tyr-Glu-Lys-Leu-Lys-Asp-Leu-Glu-Glu-GlyPGH-
Ogh ---
AGH-Phe
 HGHTyrAlaAsn-Ser-AspAsp-Leu
r 130140
BGHlle-Leu-Ala-Leu-Met-Arg-Glu-Leu-Glu-Asp-Gly-Thr-Pro-Arg-Ala-Gly-Gln-Ile-Leu-LysPGHGin-Ser
OCHVal-
ACHGin-Arg-SerGly-ProLeuArgHGHGln-ThrGly-ArgSerThr-Phe
150160
BGHGln-Thr-Tyr-Asp-Lys-Phe-Asp-Thr-Asn-Met-Arg-Ser-Asp-Asp-Ala-Leu-Leu-Lys-Asn-TyrPGHLeu
Oght-
AGHPro-Ile-His-LeuAsn-Glu ---
HGHSer-Sec-His-Asn
170180
BGHCly-Leu-Leu-Ser-Cys-Py-Arg-Lys-Asp-Leu-His-Lys-Thr-Glu-Thr-Tyr-Leu-Arg-Val-MetPGHLysAla
OGH - ACT LysValLys--
HGHTyr-ttet-AspValPhe-Ile-Val190
BGHLys-Cys-Arg-Arg-Phe-Gly-Glu-Ala-Ser-Cys-Ala-PhePGHValSer
Ogh
AGH ---. Ser-AsnThr-IleHGHGinXSer-ValtGly-Gly
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法律状态:
2005-05-10| REG| Reference to a code of a succession state|Ref country code: RU Ref legal event code: MM4A Effective date: 20040222 |

优先权:

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申请号 | 申请日 | 专利标题

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US70434185A| true| 1985-02-22|1985-02-22|

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US06704677|US4652630B1|1985-02-22|1985-02-22|

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