JPH0523195A - Purification of serum albumin - Google Patents

Abstract

PURPOSE:To purify a serum albumin produced by a gene recombination method and isolated into a microbial cultured liquid from a cultured mixture. CONSTITUTION:A serum albumin is quickly and simply separated from other protein, lipid and polysaccharide derived from a microorganism by carrying out metal chelate chromatography.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for purifying serum albumin (SA) from a microorganism culture solution or the like.

[0002]

2. Description of the Related Art SA is a plasma protein that is synthesized in large quantities in the animal body, especially in the liver, and plays an important role in maintaining physiological effects such as maintaining colloid osmotic pressure and transporting various blood proteins or low-molecular substances. There is. In recent years, SA has been used in large quantities as a drug.

For example, human serum albumin (Human Serum
Albumin, HSA) is a protein preparation that is intravenously administered to burns, nephrosis, hemorrhagic shock, etc., and its demand is extremely large.

[0004] As described above, HSA and the like, which are in great demand, are conventionally produced from human plasma, but it is necessary to produce non-plasma-derived HSA and the like due to the limitation of production amount caused by lack of raw materials and fear of pathogen contamination. Is occurring.

[0005] The gene recombination method uses such non-plasma-derived S
A method suitable for producing A is disclosed in, for example, JP-A-2-27.
6589, JP-A-3-83595, or Biotechnolog
y, 8, p 42, 1990, etc., a method for efficiently producing HSA or the like (the dose of HSA for humans is usually 5 to 10 g), which requires large-scale production using yeast as a host. Is listed.

As described above, when SA is produced by the gene recombination method, SA produced by the host microorganism is used.
Is often released into the culture solution, but at the same time, other proteins, lipids, and polysaccharides derived from the host are also released into the culture solution, and therefore an operation for purifying SA from the culture solution is required. .

Regarding the method for purifying SA, for example, a method using ethanol (Cohn et al., J. Amer. Chem. Soc. 68, p459,
1946, Kistler-Nitschmann et al., Vox Sang., 7, p 414,19.
62 Foster et al., Methods of Plasma Protein Fractionat
ion, Curling, JM, ed., Academic Press, London, p17,198
0 years), a method of combining precipitation with polyethylene glycol and various chromatographies (Hao et al., Methods
of PlasmaProtein Fractionation, Curling, JM, ed., Ac
ademic Press, London, p57, 1980), a method using an ion exchange gel having a diethylaminoethyl (DEAE) group or a carboxymethyl (CM) group (Curling et al.,
Methods of Plasma Protein Fractionation, Curling,
JM, ed., Academic Press, London, p77, 1980) or
A method by affinity chromatography using fatty acid, heparin, bull-dextran, cibacron bull, 3GA, etc. as a ligand (Takeno Banno, assembly using animal components,
Methods for purifying HSA from plasma are known, such as "Land animals", R & D Planning, p 166, 1987).

[0008]

2. Description of the Related Art As described above, the method of purifying SA is not unknown. However, these purification methods are basically methods for purifying SA in plasma, and are not methods for purifying SA produced by a microorganism by a gene recombination method from a microorganism culture solution. Since the plasma component and the microbial culture liquid component have largely different properties, the purification from the microbial culture liquid cannot be efficiently performed by the purification method from the plasma component.

Among the above-described methods for purifying SA from plasma, affinity chromatography using Cibacron blue as a ligand utilizes affinity with SA, and is also used for purifying SA from a microorganism culture solution. Expected to be useful (Travis et al., Biochem. J., 157, p301,
1976). However, the affinity-chromatography method requires the use of expensive functional groups, resulting in high cost of the purification step, and the gel carrying such functional groups needs to be strictly stored and managed. Even if the storage management of the gel is strictly performed, there are some points to be improved such that the performance is easily deteriorated.

[0010]

[Means for Solving the Problems] The inventors of the present invention have made earnest studies on a method for efficiently purifying SA produced by a microorganism by a genetic recombination method from a microorganism culture solution. The inventors have found that the objective can be achieved by using chromatography, and have completed the present invention. That is, the present invention is a metal chelate chromatography
Is a method for purifying serum albumin in a microorganism culture solution. The present invention will be described in detail below.

The present invention is a method for purifying SA produced using, for example, Escherichia coli, yeast, animal cells, etc. as a host from a culture solution containing the secretion product of the host as a contaminant. The SA purified in the present invention is not limited as long as it is produced by any host.

By the way, SA is human SA (HSA),
It is known that various SAs exist depending on animal species, such as rabbit SA (RSA, patent application filed on July 10, 1991 by the same applicant, described in reference number 911167).
However, in HSA and RSA, the prepro sequence consisting of 24 amino acid residues, the position of 35 cysteine residues, the N-terminal third His residue that is considered to be involved in binding with metal, and further the purified sample The isoelectric points are the same,
Moreover, it has a homology of 75% as a whole.
Therefore, the present invention is not limited to a method for purifying HSA, and is not limited to SA for non-HSA, such as cow, goat, rat and mouse.
Is a purification method applicable to.

The metal chelate chromatography used in the present invention is a method for purifying SA by using a gel carrying a metal and utilizing the chelate reaction between the metal and SA. There is no limitation on the base material of the gel supporting the metal, and any material that is stable in the solvent system in which it is used may be used. Examples of the supported metal include zinc, nickel, copper, cobalt, and the like. Among them, zinc can firmly bind to SA, and therefore, a gel supporting zinc is used in the present invention. It is preferable. The gel is used in a state of being packed in an appropriate column according to the method of ordinary metal chelate chromatography, but even if it is used in a beaker in some cases,
It may be used in the form of being poured into the culture solution. In this case, the gel may be separated from the culture solution as one of the natural operations for purifying SA.

In the present invention, the culture solution is adjusted to a pH of about 6.0 to 8.0 by using Tris-hydrochloric acid buffer solution, so that SA in the gel is more efficiently used as a metal-supporting gel. It can be adsorbed. In order to separate and elute the adsorbed SA from the gel, for example, an operation of adding a low molecular weight amine such as imidazole or lowering the pH may be performed. However, from the viewpoint of reproducibility, operability, separation characteristics, toxicity, etc., it is preferable to use a solution containing glycine as the separation liquid.

In the present invention, it is possible to purify SA even by using a microorganism-containing culture solution containing SA produced by the gene recombination method as a sample in contact with a metal-supporting gel. However, in order to improve the purification efficiency or to simplify the operation, it is preferable to remove the microbial cells or crushed cells by subjecting the culture solution to centrifugation, ultrafiltration, ammonium sulfate precipitation, or the like.

The purification method of the present invention can purify SA by itself, but in order to achieve higher purity purification, for example, gel filtration chromatography, ion exchange chromatography, etc. are carried out. It is preferable to use crudely purified SA as a sample, or to carry out such chromatography after the purification of the present invention. Such chromatography is not
It is a method that can be carried out rapidly without the risk of denaturing A and is suitable for the pre-treatment or post-treatment of the purification method of the invention.
Here, gel filtration chromatography is based on the molecular weight of SA, and ion exchange chromatography is based on the physical properties of SA. Therefore, as the pre-treatment or post-treatment of the present invention, these two types of chromatography
It is particularly preferred to carry out

For example, in gel filtration chromatography,
It is preferable to use one having an exclusion limit molecular weight of 100,000 or more in terms of protein and having excellent separation of proteins having a molecular weight of about 50,000 to 100,000. There is no particular limitation on the base material of the gel, but it is preferable to use a hydrophilic gel such as hydrophilic silica gel or hydrophilic vinyl polymer gel in order to prevent SA adsorption. On the other hand, in ion exchange chromatography, it is preferable to use an anion exchange gel in consideration of the physical properties of SA. Examples of such gel include diethylaminoethyl (DEAE) group and quaternary aminoethyl (QAE).
The use of gels carrying groups can be illustrated.

[0018]

EXAMPLES Examples will be described below to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 A plasmid (pYRSA3) for producing rabbit serum albumin (RSA) in yeast prepared by the same applicant as described in a patent application (reference number 91119) dated July 10, 1991, was prepared by BamHI. Linearized and spheroplast method (Cregg et al., Molec. Cell. Biol., 5, p33
76, 1985) yeast (Pichia pastoris GTS115 strain,
Japanese Patent Application Laid-Open No. 63-164891) and transformed into methano-
Le assimilating Mut ^- and the strain were prepared (JP 63-164
891).

The transformant was grown in glycerol medium, converted to methanol medium, and continued to be cultured for 5 days.
SA was produced and released in the culture medium.

Example 2 A sample obtained by centrifuging the yeast culture solution obtained in Example 1 to remove bacterial cells and crushed bacterial cells was subjected to an ultrafiltration membrane (TOSO).
The polymer substance was removed by filtration with TS-300 manufactured by KK, and further concentrated with an ultrafiltration membrane (TS-10 manufactured by Tosoh Corp.).

The concentrated sample was dialyzed against 20 mM Tris-hydrochloric acid buffer solution (pH 7.0) at 4 ° C. overnight and equilibrated with the same buffer solution (DEAE TOYOP).
EARL 650 (manufactured by Tosoh Corporation), 0 to 0.
Eluted with a 5M NaCl gradient.

The RSA elution fraction was subjected to% SDS-PAGE.
After confirming the above, this was confirmed with 50 mM NaCl and 5
A chelating column (Pharmacia, Chelating Sepharose) equilibrated with 50 mM Tris-hydrochloric acid buffer (pH 7.0) containing% (w / v) zinc acetate.
Fast Flow), washed with the same buffer, and then eluted with a gradient of 0-0.2 M glycine to elute RSA.
Was eluted.

The state of the elution fraction from the ion exchange column is shown in FIG. 1, and the state of the elution fraction from the chelate column is shown in FIG. According to FIG. 1 and FIG. 2, after the purification method of the present invention was carried out as compared with the stage (pretreatment) of purification with an ion-exchange column, the presence of impurities was not recognized, and RSA of high purity was obtained. You can see that it was done.

Example 3 The dialysate obtained in Example 2 was applied to an ion exchange column (DEAE-5PW manufactured by Toso Co., Ltd.) equilibrated with 20 mM Tris-hydrochloric acid buffer (pH 7.0), 0-0.5M
Elute with a NaCl gradient.

The elution fraction of RSA was analyzed with 10% SDS-PAG.
Confirm by performing E, which is 50 mM NaCl and 5%
(W / v) Chelate column (Tosoh) equilibrated with 50 mM Tris-hydrochloric acid buffer (pH 7.0) containing zinc acetate.
The product was applied to Chelate 5PW manufactured by Co., Ltd., washed with the same buffer, and then RSA was eluted by gradient elution of 0 to 0.14 M glycine.

For the eluted RSA fraction, a further 100
10 mM phosphate buffer (pH 7.
0) equilibrated with a gel filtration column (Tosoh Corp., G
3000 SWXL) was applied to a column in which two (3000 SWXL) were connected in series.

Further, the elution fraction from the ion exchange column, chelate column or gel filtration column was mixed with 10% SDS-P.
It was subjected to AGE, and the appearance of foreign matters was observed.

The state of the elution fraction from the ion exchange column is shown in FIG. 3, and the state of the elution fraction from the chelate column is shown in FIG.
FIG. 5 shows the state of elution from the gel filtration column, and FIG. 6 shows the results of subjecting these three elution fractions to SDS-PAGE.
Shown in. From these figures, impurities are significantly reduced after the purification method of the present invention is carried out as compared with the stage (pretreatment) purified with an ion exchange column, and further treatment with a gel filtration column (posttreatment) is carried out. From this, it was found that highly purified RSA could be obtained, and from FIG. 6, it can be seen that the finally obtained standard does not have a contaminant band.

Example 4 The yeast used in Example 1 was disrupted with a homogenizer, and this was immunized to a mouse to prepare an anti-Pichia yeast polyclonal antibody. About the three elution fractions obtained in Example 3 West
ern blotting test (Towbin et al., Proc.Natl.Acad.Sci.US
A, 76, p4350, 1979).

The results are shown in FIG. From FIG. 7, although the RSA preparation obtained by simply performing the purification operation using the ion-exchange column contains a large amount of impurities derived from yeast, the RSA preparation after purification by the chelate column of the present invention was used.
It can be seen that the preparation is a high-purity preparation from which impurities have been removed.

[0032]

INDUSTRIAL APPLICABILITY According to the present invention, SA in a microorganism culture solution can be purified quickly and easily. Compared with the conventional method for purifying SA from plasma, which was aimed at purifying SA from plasma components, the present invention provides a protein derived from a microorganism used as a host in gene recombination,
The present invention provides a method for purifying SA from lipids and polysaccharides.

Although the present invention is an efficient method for purifying SA, it is possible to provide SA of higher purity by performing ion exchange chromatography or gel filtration chromatography as pre- or post-treatment. Is. INDUSTRIAL APPLICABILITY The purification method of the present invention has a large dose of 5 to 10 g to the human body and is therefore useful when purifying HSA and the like, which must naturally remove impurities as much as possible.

[Brief description of drawings]

FIG. 1 is a chromatogram of an elution fraction from an ion exchange column in Example 2. In the figure, the elution peak
The U-shaped symbols shown above the squares indicate the fractions that were applied to the chelate column as the RSA fractions. In the figure, 0
The line starting from M shows the state of the gradient.

FIG. 2 is a chromatogram of elution fractions from the chelate column in Example 2. In the figure, the U-shaped symbol shown above the elution peak indicates the fraction obtained as the RSA fraction. Also, a line starting from 0M in the figure shows the state of the gradient.

FIG. 3 is a chromatogram of the elution fraction from the ion exchange column in Example 3. In the figure, the elution peak
The U-shaped symbol shown above the square indicates the fraction supplied to the chelate column as the RSA fraction. In the figure, 0M
The line starting with indicates the state of the gradient,
Inject indicates the start of addition of the eluate, the number below the curve showing the elution state is the time from the start of addition of the eluate, and the number above the elution peak indicates the elution time of each peak. There is.

FIG. 4 is a chromatogram of elution fractions from the chelate column in Example 3. In the figure, the U-shaped symbol above the elution peak indicates the fraction that was applied to the gel filtration column as the RSA fraction. In the figure, the line starting from 0M shows the state of the gradient.
ct indicates the start of addition of the eluate.

FIG. 5 is a chromatogram of the elution fraction from the gel filtration column in Example 3. In the figure, the U-shaped symbol shown above the elution peak indicates the fraction obtained as the RSA fraction. In the figure, the number below the curve showing the elution state indicates the time after the sample was added to the column, and the number above the elution peak indicates the elution time of each peak.

FIG. 6 is an SDS-PAG of an elution fraction from an ion exchange column, an elution fraction from a chelate column and an elution fraction from a gel filtration column in Example 3.
The result of E is shown. M is the result for the molecular weight marker, 1 is the result for the culture concentrate prepared in Example 1, 2 is the result for the elution fraction from the ion exchange column, and 3 is the result. The results are for the elution fraction from the chelate column, and the number 4 is for the elution fraction from the gel filtration column. In the figure,
The numbers on the left side of the figure indicate the molecular weights estimated from the results for the molecular weight markers, and the RSA arrow indicates the RSA band.

FIG. 7 shows the results of Example 4. In the figure, 1 is the result of the elution fraction from the ion exchange column, 2 is the result of the elution fraction from the chelate column, and 3 is the result of the elution fraction from the gel filtration column. In the figure, the numbers on the left side of the figure are the molecular weights estimated from the results of the separately performed molecular weight marker, and the arrow of RSA indicates the band of RSA.

[Procedure amendment]

[Submission date] January 17, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction content]

By the way, SA is human SA (HSA),
Rabbit SA (RSA, 1991 patent by the same applicant
Various varieties of S depending on animal species, such as Japanese Patent Application No. 194984 ).
It is known that A exists. However, HSA and R
In SA, the prepro sequence consisting of 24 amino acid residues, the positions of 35 cysteine residues, the N-terminal third His residue that is considered to be involved in binding to metal, and further the isoelectric point of the purified sample are Identical and 7 overall
It has a homology of 5%. Accordingly, the present invention is not simply stop the purification method of the HSA, such as cattle, goats, rats, mice and the like, a purification method can also be applied to the SA other than HSA.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction content]

Example 1 Rabbit serum albumin (RSA) prepared according to the description of Japanese Patent Application No. 194984, 1991 by the same applicant.
(PYRSA3) for producing yeast in yeast was linearized with BamHI, and the spheroplast method (C
regg et al., Molec. Cell. Biol. , 5,
p3376, 1985) by Yeast (Pichia
pastoris GTS115 strain, JP-A-63-16
No. 4891) and transformed with methanol
^- and the strain was prepared (JP-A-63-164891).

Claims (3)

[Claims] 1. A method for purifying serum albumin in a microbial culture solution using metal chelate chromatography. 2. The purification method according to claim 1, wherein serum albumin is adsorbed on a metal chelate gel in a Tris-hydrochloric acid buffer solution and then eluted with an eluent containing glycine. 3. The purification method according to claim 1, wherein the metal chelate chromatography uses a gel supporting zinc.