KR100443890B1 - Process for purifying human growth hormone from recombinant e. coli - Google Patents

Abstract

The present invention relates to a method for purifying human growth hormone from recombinant Escherichia coli with high yield and high purity without including a variant without a separate activation process, and (i) culturing recombinant Escherichia coli expressing human growth hormone into periplasm. After centrifugation to obtain a cell precipitate, (ii) adding a buffer solution containing sucrose to the cell precipitate, and then centrifuging to obtain a cell precipitate, and distilled water was added to the cell precipitate, followed by centrifugation to obtain periplasmic protein. Obtaining a supernatant comprising (iii) separating the supernatant by acid precipitation of the supernatant obtained in (ii), and (iv) cation exchange chromatography, anion exchange chromatography and hydrophobic interaction with the supernatant obtained in (iii). It characterized in that it comprises the step of performing the chromatography in sequence.

Description

Purification method of human growth hormone from recombinant E. coli {PROCESS FOR PURIFYING HUMAN GROWTH HORMONE FROM RECOMBINANT E. COLI}

The present invention relates to a method for purifying human growth hormone with high yield and high purity from recombinant E. coli expressing human growth hormone (hGH) in periplasm.

Human growth hormone is a protein hormone with a molecular weight of about 21,500 Daltons consisting of 191 amino acid residues, and since the extraction and purification from the human pituitary gland (Li and Papkoff, Science 124 , 1293 (1956)), much research has been conducted. For example, Beck et al. ( Science 125 , 884 (1957)) first studied the metabolic effects of human growth hormone on humans, such as MS Raben, J. Clin. Endoclinol. 18 , 901 (1958) has shown in human clinical trials that human growth hormone is effective in treating pituitary dwarfism and has been used to treat such dwarfism. Recently, human growth hormone has been used for the treatment of Turner's Syndrome, and its indication is increasing.

In spite of this usefulness, the conventional human growth hormone was obtained by a method of extracting and purifying directly from the human pituitary gland, so the yield was very limited.

Accordingly, a method of mass production of human growth hormone by genetic recombination has been attempted. Although Goddel et al. (DV Goeddel et al., Nature 281 , 544 (1979)) first produced human growth hormone by genetic recombination, it is a methionine residue at the N-terminus when compared to the native human growth hormone. Is composed of 192 amino acids to which one more is attached, which is concerned about side effects such as an increase in antibody production rate. Therefore, the development of natural human growth hormone having the same function as that expressed in the human body is required.

In order to obtain a natural human growth hormone, International Patent Publication No. WO86 / 01229 discloses a method of removing methionine by treating dipeptidyl aminopeptides I on a human growth hormone having N-terminus attached methionine. The method has the disadvantage of further adding a process for removing methionine.

In addition, European Patent Nos. 55942, 20147 and 114695 disclose methods for producing natural human growth hormone using extracellular expression in Escherichia coli, which have disadvantages of low expression levels.

In addition, a method of producing natural human growth hormone using yeast has been reported, but there is a concern of undesired glycation.

In particular, the production of human growth hormone using the intracellular production method may be contaminated by impurities derived from the host cell or culture material, it is required to go through a difficult purification process to purify high-purity pharmaceutical products. In addition, when E. coli is used as a host, most human growth hormones are insoluble and accumulate in the cytoplasm. Therefore, they are very inefficient because they must be obtained in activated form through a refolding process. Or wrong disulfide bonds, etc. are caused, and there is a difficulty in removing them again, there is a lot of loss of titer, and it is very difficult to remove unwanted variants such as misfolding. It is also produced in the form of methionine added to the N-terminus, which is the translation initiation, and it is very difficult to remove it from the production process, resulting in loss of potency and yield.

On the other hand, the production of human growth hormone using the secretion production method to periplasm can be obtained by solubilizing a natural human growth hormone without methionine at the N-terminus, and the amount of protein in the periplasm is a protein in the whole cytoplasm. Compared with less than 10% relatively less than can be much more easily purified and the cell crushing process is unnecessary, there is an advantage of minimizing the contamination of saccharides and nucleic acids in the cytoplasm. However, since protein production in periplasm is difficult to industrialize because the expression level is low, an efficient method for purifying the expressed protein with high yield and high purity is required.

It is an object of the present invention to provide a method for purifying native human growth hormone with high yield and high purity from recombinant E. coli.

1 is a photograph showing the SDS-PAGE results of the periplasm fraction according to sucrose concentration.

2 is an elution fraction obtained by performing SP-sepharose fast flow column chromatography with a supernatant after acid precipitation by adjusting the periplasm fraction to pH 5.3, and using DEAE-Sepharose fast flow (DEAE). -sepharose fast flow) SDS-PAGE photograph showing the results of column chromatography.

FIG. 3 is an SDS-PAGE photograph showing the results of performing phenyl-sepharose fast flow column chromatography with fractions eluted from DEAE-Sepharose fast flow column chromatography.

4 is a graph showing the results of performing butylsilyl silica reverse phase high pressure chromatography with purified human growth hormone.

In accordance with the above object, in the present invention, (i) culturing recombinant E. coli expressing human growth hormone into periplasm and then centrifuging to obtain a cell precipitate, (ii) a buffer solution containing sucrose in the cell precipitate After centrifugation to obtain a cell precipitate, distilled water was added to the cell precipitate, followed by centrifugation to obtain a supernatant containing a periplasmic protein, and (iii) the supernatant obtained in (ii) was acid precipitated to separate the supernatant. and (iv) performing a cation exchange chromatography, an anion exchange chromatography, and a hydrophobic interaction chromatography in sequence with the supernatant obtained in the above (iii).

Hereinafter, the present invention will be described in detail.

Recombinant Escherichia coli used in the present invention expresses human growth hormone into periplasm, and is recombinant E. coli transformed with an expression vector containing a fusion gene encoding a fusion protein in which a secretory sequence and a human growth hormone are fused. As a representative example of the recombinant E. coli, E. coli HM10011 transformed with an expression vector fused with a modified secretion sequence of E. coli thermostabilized enterotoxin II and human growth hormone described in Patent Application No. 98-38061 (KCCM-10137), HM10012 (KCCM-10138), HM10013, HM10014, HM10015, HM10016, HM10017, HM10018, HM10019 and HM10020, but are not limited thereto.

In order to express human growth hormone into the periplasm of the recombinant E. coli, the recombinant E. coli is 50 to 400 g / L of glucose as a carbon source in a fermenter containing LB medium; As inorganic salts, KH ₂ PO ₄ 2-10 g / l, (NH ₄ ) ₂ HPO ₄ 0.5-3 g / l, NaCl 1-5 g / l, MgCl ₂ 0.5-10 g / l, Inositol 0.02-0.15 g / l, 0.01 to 0.12 g / l thiamine and various trace elements; Arginine 1.5-3.5 g / l as glutaric acid, 5-20 g / l glutamic acid, 0.3-2.7 g / l tryptophan, 2.1-5.2 g / l alanine and 2.2-5.5 g / l leucine, yeast extract and bacto-tryptone Fed-batch culture can be added with additional addition. This medium composition is particularly suitable for high concentration culture of recombinant E. coli and high expression of human growth hormone to periplasm in Escherichia coli, and according to the results of the present inventors' experiment using recombinant E. coli HM10011, HM10012 and HM10013, The composition was found to significantly increase the culture concentration of recombinant E. coli, the expression level of E. coli human growth hormone, and the processing rate to periplasm. The recombinant E. coli secretes periplasm, a naturally occurring human growth hormone without the addition of additional methionine at its amino terminus. The recombinant E. coli culture thus obtained is centrifuged to obtain cell precipitates.

The cell precipitate is added with a buffer solution containing sucrose and then centrifuged to obtain a cell precipitate. Distilled water is added to the cell precipitate, followed by centrifugation to obtain a supernatant containing a periplasmic protein. In this process, periplasmic proteins, including human growth hormone, are extracted by osmotic pressure. Specifically, the cells are treated with a buffer containing sucrose, for example, a buffer containing 10% to 30% sucrose. It contracts and expands with distilled water treatment, which causes periplasmic proteins, including human growth hormone, located between the cell membrane and the cell wall to be extracted through the loosened cell wall without rupturing the cell membrane. In addition to sucrose, the osmotic extraction may use an organic solvent such as acetone and butanol. Centrifugation of the extract yields a supernatant containing periplasmic protein.

Acid is then added to the supernatant containing the periplasm protein to adjust the pH to 5.8 to 5.2, preferably 5.5 to 5.3 and centrifugation to acid precipitate the insoluble material and to obtain a supernatant containing soluble human growth hormone. As the acid used for the acid precipitation, acetic acid is preferable. In the present invention, by using a recombinant Escherichia coli secreted by human growth hormone periplasm, it is possible to extract a human growth hormone by simply obtaining a periplasm fraction without crushing the whole Escherichia coli.

Cation exchange column chromatography, anion exchange column chromatography, and hydrophobic interaction column chromatography are sequentially performed while maintaining the pH of the obtained supernatant above the isoelectric point (pI = 5.0) of human growth hormone.

In the cation exchange column chromatography, it is preferable to use a carboxymethyl (CM) -Sepharose or SP-Sepharose column, the pH of the chromatographic conditions in the range of 4 to 6, preferably in the range of 5.0 to 6.0 It is preferable to elute with a buffer solution containing acetic acid having a salt concentration of 100 mM or less while maintaining. Through such cation exchange column chromatography, nucleic acids and pigments present in the supernatant may be efficiently removed to increase purification efficiency.

It is preferable to use a DEAE-Sepharose column for the anion exchange column chromatography, and the concentration of the salt is 100 mM while maintaining the pH in the range of 5.5 to 7.5, preferably in the range of pH 5.5 to 6.6. It is preferable to elute with the following buffer solutions.

It is preferable to use a phenyl-sepharose column for the hydrophobic interaction column chromatography, and the concentration of salt is 500 while maintaining the pH in the range of 7 to 8.5, preferably in the range of pH 7.5 to 8.5. Elution with a buffer solution of no more than mM is preferred.

According to the method of the present invention, a natural human growth hormone obtained by efficiently removing variants in which demination has occurred is obtained in high yield and high purity.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Reference Example

E. coli HM10011 (KCCM-10137, Patent Application No. 98-38061) transformed with the expression vector pT14S1SH-4T22Q fused with the modified secretion sequence of E. coli thermostable enterotoxin II and human growth hormone contained 1 mg / l ampicillin The seed culture was carried out by inoculating a 2 L glass container containing 1 L of sterile LB medium (Bacterium-Tryptone 10g / L, Bacterium-Yeast Extract 5g / L, NaCl 10g / L).

The culture solution was vigorously stirred and aerated and incubated at 37 ° C. for 14 hours, followed by glucose 200 g / l as carbon source, 5 g / l KH ₂ PO ₄ as inorganic salts, (NH ₄ ) ₂ HPO ₄ 1 g / l, 2 g / l NaCl, 2 g / l MgCl ₂ , 0.07 g / l inositol, 0.05 g / l thiamine and various trace elements, yeast extract and bacto-tryptone were added during fermentation. During fermentation, dissolved oxygen (DO) was maintained at 20% or more, and 50% glucose was continuously added to ferment for the last 40 hours. After the fermentation was completed, the fermentation broth was centrifuged at 7,000 rpm to obtain the cells and stored at -70 ℃.

Example

(Step 1) Extraction of Periplasm Protein

2 kg of E. coli cells obtained in the reference example were added to 30 L of a room temperature buffer solution (20% sucrose, 1 mM EDTA, 30 mM Tris, pH 8.0), stirred for 2 hours, and the pellet was collected by continuous centrifugation at 7,000 rpm. 30 L of distilled water at 4 DEG C was added to the pellet, and the pellet was removed by continuous centrifugation at 7,000 rpm, and the supernatant containing the periplasm protein was collected. The average extraction yield was 65 ± 5% of the human growth hormone content.

In addition, the extraction yield was compared by performing the same step while decreasing the concentration of sucrose in 5%, 10% in the buffer solution. Figure 1 is a photograph showing the SDS-PAGE results of the Periplasm fraction of recombinant E. coli, where the first column is 5% sucrose, the second column is 10% sucrose, and the third column is 20% This is the case when cross is used. As shown in Figure 1, the sucrose concentration suitable for extracting the periplasmic protein from E. coli cells is 10 to 20%.

(Step 2) Acid Precipitation

1% acetic acid was added to the supernatant containing the periplasmic protein obtained in step 1 to adjust the pH to 5.3, and the insoluble matters produced at this time were removed using a continuous centrifuge at 7,000 rpm.

(Step 3) Cation Exchange Chromatography

The supernatant obtained in step 2 was subjected to SP-sepharose fast flow column chromatography as follows. The supernatant was adsorbed onto an SP-Sepharose column (200 X 110 mm, Pharmacia) pre-equilibrated with Buffer 1 (15 mM sodium acetate, pH 5.3), and the unadsorbed protein was thoroughly washed with the same buffer to remove 50 mM. Elution with buffer 1 containing sodium chloride. The eluted fractions were electrophoresed to collect fractions with a purity of at least 85% of human growth hormone.

(Step 4) Anion Exchange Chromatography

After adjusting the fractions obtained in step 3 to pH 5.8 with NaOH, DEAE-sepharose fast flow column chromatography was performed as follows. That is, the fractions were adsorbed onto a DEAE Sepharose column previously equilibrated with Buffer 2 (20 mM sodium acetate, pH 5.8), and then eluted with increasing sodium chloride concentration by 10 mM to elute human growth hormone. SDS-PAGE was performed using the eluted fractions, followed by staining with coomassie blue, and fractions having a purity of 97% or more of human growth hormone were collected. FIG. 2 shows SDS-PAGE results of the elution fractions obtained by DEAE-Sepharose fast flow column chromatography, in which the first column is a standard and the second column is an elution fraction.

(Step 5) Hydrophobic Interaction Column Chromatography

After adjusting the pH of the human growth hormone solution obtained in step 4 to 8.0 and adding sodium chloride to a final concentration of 1M, phenyl-sepharose fast flow column chromatography was performed as follows. That is, the solution was adsorbed onto a phenyl-sepharose column (Pharmacia Co., Ltd.), which had been previously equilibrated with a buffer solution 3 (10 mM Tris, pH 8.0, 1M sodium chloride) at a flow rate of 4 l / hour, and then the column was buffered with buffer 3. Human growth hormone was eluted with buffer 4 (10 mM Tris buffer solution, pH 8.0, 0.5 M sodium chloride) while absorbance was washed until the absorbance was less than 0.05 at 280 nm and the sodium chloride concentration was gradually decreased. The eluted fractions were electrophoresed to collect fractions of 99% or higher purity of human growth hormone to obtain 3.4 g of human growth hormone. FIG. 3 shows the SDS-PAGE results of the fractions obtained by phenyl-sepharose fast flow column chromatography, where the first column is the sample before purification and the second column is the final purified sample.

The N-terminal sequence of the purified human growth hormone was confirmed to be the same as the purified human growth hormone in the human pituitary gland, and the endotoxin was 1 EU / IU hGH or less.

Test Example

(1) in vivo titer analysis

In order to investigate the physiological activity of the recombinant human growth hormone of the present invention, 6 weeks old pituitary gland rats were injected with 20 μg / day and 40 μg / day of purified human growth hormone and international standard (NIBSC), respectively. After administration to (Hyposectomized Sprague Dawley rat, body weight 80-100 g, Japanese SLC), the weight change was measured. At this time, the control was carried out in the same manner using physiological saline. As a result, the body weight of rats to which the recombinant human growth hormone of the present invention and the international standard were administered was significantly increased and showed a dose-dependent tendency to increase. In addition, rats administered the recombinant human growth hormone of the present invention gained weight similar to that of the international standard.

As a result of visual observation of pituitary gland extraction, the remaining pituitary gland was not observed in all rats, which confirms the reliability of the experimental results.

In summary, as a result of comparing the physiological activity of the human growth hormone of the present invention on the basis of an international standard, it was 3.0 IU / mg or more.

(2) variant analysis

Purified human growth hormone in step 5 of the Example using a butylsilyl silica column according to the method of the European Pharmacopia 3rd Edition 1203-1204 and 29% propanol and 71% 50 mM Tris buffer solution (pH 7.5) as the mobile phase Reversed phase high pressure column chromatography was carried out using the results shown in FIG. 4. As shown in Figure 4, it can be confirmed that the variant of human growth hormone, which is very similar in physical physicochemical properties to normal human growth hormone, such as delamination in the human growth hormone of the present invention was efficiently removed.

According to the method of the present invention, a natural human growth hormone having the same function as that expressed in the human body in a simple, high yield and high purity can be obtained without a separate activation process. In particular, according to the method of the present invention, variants of human growth hormone expressed in E. coli are efficiently removed.

Claims (8)

(i) culturing recombinant E. coli expressing human growth hormone into periplasm and then centrifuging to obtain cell precipitates; (ii) adding a buffer solution containing sucrose to the cell precipitate followed by centrifugation to obtain a cell precipitate, and adding distilled water to the cell precipitate and centrifuging to obtain a supernatant containing periplasmic protein; (iii) separating the supernatant by acid precipitation of the supernatant obtained in (ii); (iv) the supernatant obtained in (iii) above with cation exchange chromatography maintained at pH 5.0-6.0, anion exchange chromatography maintained at pH 5.5-6.6, and hydrophobic interaction chromatography maintained at pH 7.5-8.5 Steps to Perform Graphics Characterized in that the purification method of human growth hormone. The method of claim 1, The recombinant E. coli is HM10011 (KCCM-10137) or HM10012 (KCCM-10138). The method of claim 1, Using an SP-Sepharose column in the cation exchange chromatography. The method of claim 3, wherein The cation exchange chromatography is performed by eluting with a buffer containing acetic acid. The method of claim 1, Using DEAE-Sepharose column in the anion exchange chromatography. The method of claim 5, wherein The anion exchange chromatography is performed by eluting with a buffer containing acetic acid. The method of claim 1, Using a phenyl-sepharose column in said hydrophobic interaction chromatography. delete