JP2001321193A - Method for roughly purifying human growth hormone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for roughly but efficiently purifying a human growth hormone from the liquid extracted from microbial biomass containing the human growth hormone. SOLUTION: This method comprises the steps of adding low-molecular chitosan to the liquid extracted from microbial biomass capable of producing the human growth hormone to form aggregates to separate them, adding a salt of a transition metal to the obtained solution to form an insoluble complex in the solution, and collecting the complex.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for simply and efficiently purifying hGH from an extract of cells of a microorganism that produces human growth hormone (hGH). hGH is a useful substance used as a therapeutic agent for pituitary insufficiency short stature and the like.

[0002]

2. Description of the Related Art Growth hormone is a typical peptide hormone secreted from the anterior pituitary gland and has a molecular weight of about 22%.
Two types are known, one of 000 and about 20,000. This growth hormone has various organisms, has a different structure for each organism, and has high biological species specificity. Although growth hormone has a wide range of physiological actions, it is well known that it has an action of binding to a growth hormone receptor and exhibiting a growth promoting activity.

On the other hand, in recent years, with the advance of recombinant DNA technology, a large amount of microorganisms transformed by using a plasmid into which a gene of a physiologically active substance or a useful protein existing in a living body is incorporated has been developed. It has become possible to produce useful substances. In the case of hGH, a recombinant microorganism obtained by connecting the DNA sequence of hGH to an appropriate promoter, incorporating this sequence into an expression vector and transforming a host microorganism is cultured in an appropriate medium, It has become possible to produce hGH in the cells or in the periplasmic space between the outer and inner membranes.

In order to purify hGH from such a recombinant microorganism, the following method is generally employed. That is, first, a large amount of a recombinant microorganism producing hGH is cultured,
The cells are collected from the culture by centrifugation or the like, and the cells are collected by freeze-thawing, ultrasonic crushing, osmotic shock, or the like, to destroy the cell wall or cell membrane of the microorganisms, and the microorganisms together with hGH are disrupted. An extract containing a large amount of various proteins derived from the extract is obtained. Further, cell residues are removed from the extract by centrifugation or membrane separation, and then various separation modes such as a combination of column chromatography such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, hydrophobic chromatography, and reverse phase chromatography. As a result, the target substance h
This is a method for purifying GH.

In purifying hGH by a method combining column chromatography, it is necessary to efficiently remove impurity proteins other than hGH. However, the extract of microbial cells containing hGH usually contains a large amount of proteins derived from various types of microbial cells.
These are impure proteins other than the target hGH. Many of these impurity proteins adsorb to the chromatographic carrier. Since the adsorption capacity of the chromatographic carrier is fixed, if the amount of impurity protein adsorbed is large, the adsorbing capacity of hGH is relatively reduced, and as a result, the amount of the chromatographic carrier required is inevitably increased, and the column size is also increased. It will be bigger.

In general, various buffers used for column chromatography, water for column washing or column regeneration,
Ethanol, alkali solution, salt solution and the like are used in an amount proportional to the volume of the chromatographic carrier. Therefore,
When the column size becomes large, the amount of the solution used inevitably increases, and as a result, the raw material cost increases and hG
The manufacturing cost of H will increase. Also, if the amount of buffer solution etc. to be used increases, naturally,
It is inevitable that the volume of the equipment for preparing the buffer solution and the like and the container for storing the prepared buffer solution and the like also increase, and the amount of money required for the associated equipment also increases.

Means for solving such a problem include:
Before the first column chromatography, h
It is conceivable that the purity of hGH is further improved by performing the crude purification of GH.
The subsequent purification step can be simplified. That is, if the number of column chromatography can be reduced by performing the crude purification, the production cost is reduced, which is a very effective means. However, when the recovery rate of hGH in the crude purification is low, the production cost of hGH also increases.
A method that satisfies the two conditions of increasing the purity of H and recovering hGH with high yield is desired.

In general, in crude purification of a protein, salting out utilizing a difference in solubility of the protein and fractional precipitation using an organic solvent are often used. However, when these general methods are applied to roughly purify hGH from an extract of the cells of a microorganism as described above, the recovery rate decreases if high purity is to be obtained, In addition, when the recovery rate is emphasized, there is a disadvantage that the purity of the obtained hGH is reduced. Therefore, there is a problem in employing the above method as an industrial method for producing hGH. At present, there is no known crude purification method that can fundamentally solve such a problem.

On the other hand, as a similar technique in this field, Japanese Patent Application Laid-Open No. 63-216898 discloses a method for recovering somatotropin from a dilute aqueous solution containing somatotropin (growth hormone having a molecular weight of about 22,000). I have. The disclosure relates not to a crude purification method but to a method for recovering somatotropin from a dilute aqueous solution containing only purified somatotropin.

That is, Japanese Patent Application Laid-Open No. Sho 63-216898 discloses that a dilute aqueous solution containing only somatotropin obtained as a result of purifying somatotropin from a pituitary homogenate or a fermentation broth is prepared. A method for recovering somatotropin by adding a salt and separating the generated insoluble complex of somatotropin and a transition metal, and recovering somatotropin from a microbial cell extract containing various impurity proteins as described above. Not a way.

[0011] However, the method disclosed in the above-mentioned publication discloses a method for extracting hGH from microbial cell extracts containing various impure proteins.
The present inventors have considered that the method may be effective as a method for recovering the microorganisms. Therefore, the present inventors have attempted the method described in JP-A-63-216898 for an extract of the microbial cells containing hGH. Was. That is, a transition metal salt was added to an extract of microbial cells containing hGH, and it was attempted whether or not hGH could be roughly purified. However, as shown in Examples described later, particularly Comparative Example 1,
When this method is performed on an extract of microbial cells containing hGH, the purity of hGH is hardly improved, and the recovery rate is less than 70%. I knew it wasn't.

[0012]

DISCLOSURE OF THE INVENTION The present invention provides a method for efficiently purifying hGH from an extract of cells of a microorganism containing hGH, that is, a method for recovering high-purity hGH with a sufficient yield. It is intended to provide.

[0013]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object. As a result, first, an extract of microbial cells containing hGH was treated with low-molecular-weight chitosan, and the resulting aggregates were removed. When a transition metal salt is subsequently added to this solution after separation, it has been found that hGH can be recovered extremely efficiently, and that its purity is dramatically improved.
The present invention has been completed.

That is, according to the present invention, a low molecular weight chitosan is added to an extract of cells of a microorganism producing hGH to form an aggregate, which is then separated.
A method for crude purification of hGH, comprising: adding a transition metal salt to the obtained solution to form an insoluble complex in the solution; and recovering the complex. It is a method for rough purification, wherein the transition metal salt is a zinc salt or a copper salt.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The hGH referred to in the present invention is hGH produced by a recombinant microorganism and has a molecular weight of about 200.
00, or about 22,000 hGH.

[0016] As the microorganism in the present invention, a recombinant microorganism producing hGH is used. Such a recombinant microorganism can be generally obtained by connecting the hGH DNA sequence to an appropriate promoter sequence, incorporating this sequence into an expression vector, and transforming a host microorganism. The host microorganism is not particularly limited, but Escherichia coli, Bacillus subtilis and the like are generally used.

In the present invention, the extract of the cells of the microorganisms that produce hGH refers to a culture of a recombinant microorganism that produces hGH, and then collecting the cells by a method such as centrifugation or membrane separation. Next, it refers to a liquid obtained by destroying the cell wall or cell membrane of a microorganism by freeze-thawing, sonication, or osmotic shock.

The low molecular weight chitosan used in the present invention is a low molecular weight chitosan obtained from natural chitin, and has an intrinsic viscosity of 0.01 to 5 (dl / g), preferably 0.03 to 2 (dl / g). (Dl / g), for example, those described in Japanese Patent Publication No. 6-41479. Also,
Such a low-molecular-weight chitosan is commercially available, for example, as Cream Remover I (trade name, manufactured by Kurita Industry Co., Ltd.), and is useful in the present invention.

In the present invention, the low-molecular-weight chitosan may be added in the form of a powder as it is.
It is preferable to add as a 0% (w / v) aqueous solution.

The above aqueous solution of low molecular weight chitosan is generally prepared by the following method. That is, first, the required amount of low molecular weight chitosan is suspended in water, and hydrochloric acid,
The low molecular weight chitosan is dissolved by adding an aqueous solution such as acetic acid. Then, water is added and the final volume is adjusted to prepare a low-molecular-weight chitosan solution having a desired concentration.
Before adjusting the final volume, it is preferable to adjust the pH of the low molecular weight chitosan solution to 4 to 6 by adding an aqueous alkali solution such as sodium hydroxide or sodium carbonate.

In the present invention, the low molecular weight chitosan solution prepared as described above is added little by little after adjusting the extract of the microbial cells containing hGH to an appropriate pH, preferably pH 6 to 8.

The low molecular weight chitosan is usually 0.02% (W /
V) It is added so as to have a concentration of not less than. The amount of the low molecular weight chitosan added is proportional to the amount of cell residue, nucleic acid, endotoxin, etc. contained in the cell extract. These contents vary depending on the type of the recombinant hGH producing microorganism. When an excessive amount of low-molecular-weight chitosan is added, the recovery rate of hGH may decrease with a decrease in pH or the like. Therefore, it is desirable to determine the amount of the low molecular weight chitosan in advance so that the amount of the low molecular weight chitosan is minimized. For example, an appropriate addition amount is determined by the following method. That is, after a small amount of the cell extract is collected and adjusted to an appropriate pH, chitosan is added in various stepwise amounts. Next, aggregates are removed by a method such as centrifugation to obtain a chitosan-treated extract.
The turbidity of this chitosan-treated extract or the minimum amount of addition at which the absorbance at 260 nm (the wavelength at which the absorption of nucleic acids is maximum) is at a minimum level is determined. This point is the minimum necessary amount of low-molecular-weight chitosan that can remove cell residue, nucleic acid, endotoxin, etc. contained in the cell extract. Next, based on this, a large-volume extract may be treated by adding a necessary minimum amount of low-molecular-weight chitosan.

In the present invention, when low-molecular-weight chitosan is added, cell residues, nucleic acids, endotoxin, etc., which cannot be completely removed by a method such as centrifugation, of the microbial cell extract, bind to the low-molecular-weight chitosan and become insoluble. Agglomerates form and begin to precipitate. Aggregates formed in this manner can be relatively easily removed by a method such as centrifugation or separation using a microfiltration membrane.

Next, in the present invention, a salt of a transition metal is added to the solution obtained as described above while stirring the solution. By adding a transition metal salt, a precipitate containing an insoluble complex of hGH can be generated in the solution.

In the present invention, zinc or copper is preferred as the transition metal. The form of these salts is not particularly limited, and specific examples thereof include sulfates, acetates, nitrates, and hydrochlorides. These transition metal salts can be added in the form of a powder, but are preferably added little by little as an aqueous solution.

The transition metal salt is usually added to a concentration of 0.5 mM or more. However, the required amount of addition is affected by the content of hGH in the chitosan-treated extract and the type and amount of the impure protein. If the addition amount is too small, the recovery rate of hGH will decrease. On the other hand, if the amount of addition is excessive, a large amount of impurity proteins other than hGH will precipitate, and the purity will decrease. Therefore, preliminary
The transition metal salt is added to the chitosan-treated extract in various stepwise amounts and the mixture is roughly purified. In consideration of the above, it is desirable to determine an appropriate range of the addition amount.

In the present invention, when the insoluble complex containing hGH formed as described above is allowed to stand for a while, almost all of the insoluble complex sediments. However, centrifugation, separation using a microfiltration membrane, or filtration. And the like, the entire amount of the insoluble complex can be easily recovered.

The precipitate containing hGH recovered as described above may be recovered immediately after recovery or frozen and stored at -10 ° C. or lower, and then subjected to an acidic or alkaline buffer, ethylenediaminetetraacetic acid or the like. By adding a chelating agent of the formula (1), it can be easily redissolved.

[0029]

EXAMPLES The present invention will be described in detail below, but the present invention is not limited by these examples.

Comparative Example 1 Recombinant Escherichia coli producing hGH having a molecular weight of about 20,000 described in JP-A-8-322586 (Deposited number: FERMBP-5, Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology)
020) was cultured in a medium consisting of peptone, yeast extract, glycerol, succinic acid and tetracycline. The cultured cells were collected by centrifugation, and an extract was prepared by an osmotic shock method.

For 90 ml of this cell extract, 200 m
9 ml of M Tris-HCl buffer (pH 7.5) was added, and the extract was adjusted to pH 7.5 with 3N sodium hydroxide. This p
20 ml of the H-adjusted extract was stirred with a magnetic stirrer while stirring 2 ml of 500 mM ZnSO ₄ .
Was added dropwise to form a precipitate. After the solution was allowed to stand at 4 ° C. for 1 hour, the precipitate was collected by centrifugation.

To the obtained precipitate, 20 ml of 10 mM Tris-HCl buffer (pH 8) and 2 ml of 100 mM ethylenediaminetetraacetic acid (pH 8) were added to dissolve the precipitate.
A crude liquid was obtained.

The hGH concentration of the extract and the crude solution was measured by an enzyme immunoassay. Further, the absorbance at 280 nm was measured as an index indicating the total protein amount. Table 1 shows the results.

[0034]

[Table 1]

Example 1 By the same method as described in Comparative Example 1,
No. 586, an extract of recombinant Escherichia coli producing hGH having a molecular weight of about 20,000 (Deposit No. FERM BP-5020, National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology) was prepared.

For 100 ml of the cell extract, 200
10 ml of mM Tris-HCl buffer (pH 7.5) was added, and the pH was adjusted to 7.5 with 3N sodium hydroxide. While stirring the pH-adjusted extract with a magnetic stirrer,
5.5 ml of an aqueous solution (pH 4) of 5% low-molecular-weight chitosan (manufactured by Kurita Kogyo Co., Ltd., trade name: pH 4) was added dropwise to form aggregates, and stirring was continued for 10 minutes. Then, the aggregate was removed by centrifugation to obtain a chitosan-treated extract.

Four 20 ml of this chitosan-treated extract were weighed, and each of them was stirred with a magnetic stirrer to obtain 500 mM ZnSO ₄ , ZnO ₄ .
Any 2 ml of (CH ₃ COO) ₂ , Zn (NO ₃ ) ₂ , or CuSO ₄ was added dropwise to form a precipitate. After leaving each liquid at 4 ° C. for 1 hour, the precipitate was collected by centrifugation.

To the resulting precipitate, 20 ml of 10 mM Tris-HCl buffer (pH 8) and 0.5 ml of 100 mM ethylenediaminetetraacetic acid (pH 8) were added, and the precipitate was dissolved to obtain a crude purified solution.

The hGH concentrations of the extract and the crude solution were measured by an enzyme immunoassay. In addition, the absorbance at 280 nm was measured as an index indicating the total protein amount. Table 2 shows the results.

From the results shown in Table 2, the extract of hGH-producing microorganism cells was extracted from the extract of hGH using the method of the present invention.
It can be seen that in the case of crude purification, both the recovery rate and purity of hGH are greatly improved as compared with Comparative Example 1.

[0041]

[Table 2]

Example 2 A molecular weight of about 200 described in Japanese Patent Application No. 11-194510.
00 hGH-producing recombinant Escherichia coli (Deposit No. FERM BP-629, National Institute of Biotechnology and Industrial Technology)
0) was cultured in a medium consisting of peptone, yeast extract, glycerol, succinic acid and tetracycline. The cultured cells were collected by centrifugation, and an extract was prepared by an osmotic shock method.

For 100 ml of this cell extract, 200
10 ml of mM Tris-HCl buffer (pH 7.5) was added, and the pH was adjusted to 7.5 with 3N sodium hydroxide. While stirring the pH-adjusted extract with a magnetic stirrer,
2.64 ml of an aqueous solution (pH 4) of 5% low-molecular-weight chitosan (manufactured by Kurita Kogyo Co., Ltd., trade name: pH4) was added dropwise to form aggregates, and stirring was continued for 10 minutes. Then, the aggregate was removed by centrifugation to obtain a chitosan-treated extract.

While stirring 20 ml of this chitosan-treated extract with a magnetic stirrer, add 500 mM
Of ZnSO ₄ was added dropwise to form a precipitate. After the solution was allowed to stand at 4 ° C for 2 hours, the precipitate was collected by centrifugation and stored at -20 ° C.

Two days after the above, the precipitate stored at -20 ° C was taken out and added to a 20 mM borate buffer (pH
9) in 19 ml and 100 m ethylenediaminetetraacetic acid (p
H9) was added in an amount of 2 ml to dissolve the precipitate to obtain a crudely purified liquid.

The hGH concentrations of the extract and the partially purified solution were measured by an enzyme immunoassay. In addition, the absorbance at 280 nm was measured as an index indicating the total protein amount. Table 3 shows the results. From the results shown in Table 3, using the method of the present invention, the extract of hGH-producing microorganism
When H is roughly purified, it can be seen that both the recovery rate and purity of hGH are greatly improved as compared with Comparative Example 1.

[0047]

[Table 3]

[0048]

As described above, when the method of the present invention is employed, the extract of the cells of the microorganism that produces hGH
It is possible to precipitate hGH with very good yield and high purity, and therefore it is a very effective method for roughly purifying hGH.

Claims (3)

[Claims] 1. A low molecular weight chitosan is added to an extract of a cell of a microorganism that produces human growth hormone to form an aggregate, which is separated, and then a transition metal salt is added to the obtained solution. A crude purification method of human growth hormone, comprising forming an insoluble complex in a liquid and recovering the complex. 2. The method according to claim 1, wherein the microorganism is a recombinant Escherichia coli. 3. The method according to claim 1, wherein the transition metal salt is a zinc salt or a copper salt.