JPH10101696A - Removal of contaminants included in protein expressed in transformant and purified protein therefrom - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the process which can separate and remove contaminants included in the substances containing proteins expressed by the transformant obtained by the genetic recombination technology and can simply and inexpensively obtain a protein of high purity without much troublesome and complicated steps or not through multi-stage process having many steps and provide a protein of high purity freed from contaminants prepared by the process. SOLUTION: A substance containing a protein expressed by a transformant obtained by the genetic recombination technology is heated in a buffer solution with a pH of 5.5-7.0 at 55-85 deg.C to separate the precipitation formed by this treatment whereby the contaminants included in the substance containing a protein formed by the gene expression of a transformant are removed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for easily removing contaminants contained in a protein-containing substance expressed in a transformant obtained by a genetic recombination technique and a method for removing contaminants by the method. It relates to proteins from which substances have been removed. In particular, the present invention is suitable for a core antigen of hepatitis B virus expressed in a transformant and the like. The present invention is particularly useful in the life science fields such as the medical field and the clinical test field.

[0002]

2. Description of the Related Art Hepatitis B virus (HBV) causes acute hepatitis due to infection of humans, and it is suspected that a part of the disease will shift from chronic hepatitis to cirrhosis and liver cancer. It is known that carriers become carriers due to vertical infection and horizontal infections occur by blood transfusion. The use of such hepatitis B virus-contaminated blood for blood transfusion causes fulminant hepatitis, which is highly likely to result in death. B in the blood
The presence of hepatitis B virus has been conventionally diagnosed by measurement of hepatitis B surface antigen (HBs antigen) detected in serum samples, but anti-hepatitis B virus core antigen can be detected in serum in which HBs antigen is not detected. If an antibody (anti-HBc antibody) is detected, it indicates that hepatitis B virus may be present. Therefore, the measurement of anti-HBc antibody is an effective means for removing transfused blood contaminated with hepatitis B virus.
As a material for measuring Bc antibodies, large-scale purification of hepatitis B virus core antigen (HBc antigen) is required.

Hitherto, methods for purifying proteins such as HBc antigen expressed in a transformant obtained by a genetic recombination technique have been reported as a method combining density gradient centrifugation and gel filtration. In order to obtain a product having satisfactory purity, a multi-stage operation such as ion exchange chromatography, sucrose density gradient centrifugation, cesium chloride (CsCl) centrifugation, and gel filtration is required, and gel filtration is required. Requires a great deal of labor and time, for example, because only substances containing a small amount of protein can be processed at one time. (Literature: M. Roggendorf et al., J. Virol.
official Methods, 6, 61, 198.
3 years, B. Frank et al. Virology, 64
Therefore, under such circumstances, it is desired to establish a means for purifying, obtaining, and supplying a protein expressed in a transformant in a large amount and in a simple manner with high purity under such circumstances. I was

[0004]

When a target protein is obtained from a transformant obtained by a genetic recombination technique, a contaminant derived from a host used in the genetic recombination operation, for example, a substance other than the target protein is used. It has been an important issue to remove host substances and other contaminants such as proteins to obtain high-purity proteins.However, conventional methods require sufficient removal of host contaminants by simple operations. In order to obtain a protein of high purity without containing contaminants, it was necessary to go through a very complicated and complicated process as described above and a multi-step process, which required a great deal of labor and time. It was difficult to apply industrially.

[0005] The inventors of the present invention have conducted intensive studies with the aim of solving the problems in obtaining a protein from a transformant obtained by this genetic recombination technique, and as a result, the substance containing the protein has a pH of 5.5. It has been found that by heating in a buffer within the range of pH 7.0 to 55 ° C. to 85 ° C., host-derived contaminants can be separated and removed, which is a very troublesome and complicated process. Without passing through multiple steps, it is possible to separate and remove contaminants contained in a substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
A method for obtaining a high-purity protein in a large amount, simply and at low cost, and a high-purity protein from which contaminants have been removed by this method have been completed.

[0006]

The present invention provides the following inventions.

(1) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is converted into a buffer solution having a pH of 5.5 to 7.0 in a temperature range of 55 ° C. to 85 ° C. A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating the mixture in a reactor and separating and removing a precipitate generated thereby.

(2) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is treated in a buffer solution having a pH of 5.5 to 7.0 in a temperature range of 60 ° C. to 80 ° C. A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating the mixture in a reactor and separating and removing a precipitate generated thereby.

(3) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is converted into a buffer solution having a pH of 6.0 to 6.5 in the range of 55 ° C. to 85 ° C. A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating the mixture in a reactor and separating and removing a precipitate generated thereby.

(4) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is treated with a buffer solution having a pH of 6.0 to 6.5 in the range of 60 ° C. to 80 ° C. A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating the mixture in a reactor and separating and removing a precipitate generated thereby.

(5) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is converted into a buffer solution having a pH of 5.5 to 7.0 in a temperature range of 55 ° C. to 85 ° C. A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating the mixture in a reactor, separating and removing a precipitate formed thereby, and performing a hydroxyapatite chromatography treatment.

(6) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is treated with a buffer solution having a pH of 5.5 to 7.0 in a temperature range of 60 ° C. to 80 ° C. A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating the mixture in a reactor, separating and removing a precipitate formed thereby, and performing a hydroxyapatite chromatography treatment.

(7) One or more steps selected from ultrasonic crushing, enzymatic treatment, surfactant treatment and ammonium sulfate fractionation from a transformant obtained by a genetic recombination technique, wherein the protein-containing substance is obtained by a genetic recombination technique. The method for removing contaminants according to the above (1), (2), (3), (4), (5) or (6), which is obtained through:

(8) A substance containing the core antigen of hepatitis B virus expressed in the transformant obtained by the genetic recombination technique is added to a buffer solution having a pH of 5.5 to 7.0 in a buffer solution having a pH of 55 to 55. Removing the contaminants contained in the substance containing the core antigen of hepatitis B virus expressed in the transformant, by heating in the range of from 85 ° C. to 85 ° C. and separating and removing the precipitate formed thereby. Method.

(9) A substance containing a core antigen of hepatitis B virus expressed in a transformant obtained by a genetic recombination technique is added to a buffer solution having a pH of 5.5 to 7.0 in a buffer solution. Removing the contaminants contained in the substance containing the core antigen of hepatitis B virus expressed in the transformant, by heating in the range of from 80 ° C to 80 ° C and separating and removing the precipitate formed by the heating. Method.

(10) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is converted into a buffer solution having a pH of 5.5 to 7.0 in the range of 55 ° C. to 85 ° C. A protein from which contaminants have been removed by heating in a vessel and separating and removing a precipitate formed thereby.

(11) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is subjected to a buffer solution at a pH of 5.5 to 7.0 in a temperature range of 60 ° C. to 80 ° C. A protein from which contaminants have been removed by heating in a vessel and separating and removing a precipitate formed thereby.

(12) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is immersed in a buffer solution having a pH of 6.0 to 6.5 in a temperature range of 55 ° C. to 85 ° C. A protein from which contaminants have been removed by heating in a vessel and separating and removing a precipitate formed thereby.

(13) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is converted into a buffer solution at a pH of 6.0 to 6.5 in the range of 60 ° C. to 80 ° C. A protein from which contaminants have been removed by heating in a vessel and separating and removing a precipitate formed thereby.

(14) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is subjected to a buffer solution at a pH of 5.5 to 7.0 in the range of 55 ° C. to 85 ° C. A protein from which contaminants have been removed by heating in a reactor, separating and removing the resulting precipitate, and performing a hydroxyapatite chromatography treatment.

(15) A substance containing a protein expressed in a transformant obtained by a genetic recombination technique is subjected to a buffer solution at a pH of 5.5 to 7.0 in a temperature range of 60 ° C. to 80 ° C. A protein from which contaminants have been removed by heating in a reactor, separating and removing the resulting precipitate, and performing a hydroxyapatite chromatography treatment.

(16) One or more steps selected from sonication, enzyme treatment, surfactant treatment and ammonium sulfate fractionation from a transformant obtained by a genetic engineering technique, wherein the protein-containing substance is obtained by a genetic recombination technique. (10), (11), (12), (1)
3) The protein according to (14) or (15).

(17) A substance containing a core antigen of hepatitis B virus expressed in a transformant obtained by a genetic recombination technique is added to a buffer solution having a pH of 5.5 to 7.0 in a buffer solution having a pH of 55 to 55. A hepatitis B virus core antigen from which contaminants have been removed by heating at a temperature in the range of from 0 ° C to 85 ° C, and separating and removing the resulting precipitate.

(18) The substance containing the core antigen of hepatitis B virus expressed in the transformant obtained by the genetic recombination technique is added to a buffer solution having a pH of 5.5 to 7.0 in a buffer solution having a pH of 5.5 to 7.0. A hepatitis B virus core antigen from which contaminants have been removed by heating at a temperature in the range of 80 ° C to 80 ° C, and separating and removing the resulting precipitate.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a substance containing a protein expressed in a transformant obtained by a genetic recombination technique refers to a gene sequence encoding an amino acid sequence of a protein to be obtained. A vector, which is then transferred to a host such as a bacterium, animal cell or plant cell to prepare a transformant, which is cultured, and a substance containing the protein of interest extracted from the transformant or Acquisition of a substance containing the protein of interest secreted into the culture broth or a substance containing the protein of interest which has been further separated or crudely purified and expressed in a transformant obtained by genetic recombination technology Is a substance containing a protein of interest.

In the present invention, the protein is not particularly limited as long as it is expressed in a transformant obtained by a genetic recombination technique.

Examples of the protein include antigens in microorganisms such as bacteria and viruses. More specifically, hepatitis B virus surface antigen (H
Bs antigen), core antigen (HBc antigen) or HBe antigen, hepatitis A virus antigen, hepatitis C virus antigen, D
Hepatitis virus antigen, hepatitis E virus antigen, hepatitis G virus antigen, human immunodeficiency virus antigen (HIV antigen), adult T cell leukemia virus antigen (ATLV antigen), Treponema pallidum antigen (TP antigen) or chlamydia Examples of the antigen include an antigen and the like, and a core antigen (HBc antigen) of hepatitis B virus is particularly preferable.

In the present invention, the antigen includes all of the antigen, part of the antigen, and antigen particles which are an aggregate of the antigen.

The transformant obtained by the genetic recombination technique of the present invention is described in "D. Hanahan, J. et al.
Mol. Biol. 166, 557, 1983.
For example, it can be prepared by a known method described in “Year” and the like, for example, by the following procedure. By inserting all or a part of the gene sequence of the protein to be obtained into the cloning site of the expression plasmid cleaved with an appropriate restriction enzyme, and ligating it using DNA ligase, Construct an expression vector. The expression vector of this protein is transferred to a host such as a bacterium, an animal cell or a plant cell by a known method such as electroporation or host competition,
Prepare a transformant.

The method for preparing this transformant will be described below, taking as an example the case of expressing HBc antigen. Hepatitis B virus (HBV) DNA as a template,
Using primers incorporating NcoI and HindIII restriction sites, polymerase chain reaction (P
A gene fragment containing the entire gene sequence of the HBc antigen is amplified by the CR method). This gene fragment was digested with the restriction enzymes NcoI and HindII.
I, digested with NcoI, HindIII
Into a cloning site of an expression plasmid pET system (manufactured by Novagen) digested with <1> to obtain an expression vector pGHBc. The expression plasmid obtained above was transformed into E. coli BL21.
(DE3), "D. Hanahan, J. Mol. Bi.
ol. 166, 557, 1983 ”and the like, and a transformant having an HBc antigen-producing ability can be obtained.

The transformant obtained by the gene recombination technique of the present invention is cultured in an LB medium or the like according to a known method. Then, the transformant and the culture solution are separated by, for example, centrifugation or filtration of the culture, and the target protein is extracted from the transformant or the transformant containing the substance containing the target protein. A culture solution from which the contained substance is secreted can be obtained.

When a protein-containing substance is extracted from the transformant, the transformant is suspended in a buffer or the like and then subjected to a treatment such as sonication, an enzyme treatment or a surfactant treatment, or a mixture thereof. The transformant is crushed by a combination of treatments, followed by centrifugation and fractionation of the supernatant, whereby the substance containing the target protein can be extracted and obtained. Further, these substances containing the protein secreted in the extraction or culture solution are subjected to ammonium sulfate fractionation treatment, and the resulting precipitate is further separated or crudely purified by suspending the precipitate in a buffer or the like. May be performed.

The protein-containing substance obtained as described above is suspended in a buffer solution having a pH in the range of 5.5 to 7.0. The buffer used for this buffer is pH 5.5 to pH 5.5.
Any substance having a buffering capacity within the range of 7.0 can be used without any particular limitation.

Examples of the buffer include MES, B
is-Tris, ADA, ACES, PIPES, MO
PSO, MOPS, BES, HEPES or TES
Good's buffer such as, for example, organic buffers such as citric acid, disodium citrate, sodium hydrogen succinate, sodium hydrogen maleate or potassium hydrogen phthalate, or inorganic such as boric acid, phosphoric acid or imidazole System buffers and the like.

The buffer may be used not only in one kind but also in combination of two or more kinds.

The concentration of the buffer in the buffer may be appropriately selected within a range having a sufficient buffering capacity in the buffer used, and generally, it may be 10 mM or more.

The pH of this buffer is adjusted to pH 5.5 to pH 7.5.
By setting the pH within the range of 0, denaturation due to heat during the heat treatment of the protein to be obtained can be prevented.
It is more preferable to set H in the range of pH 6.0 to pH 6.5 because the stability can be further increased.

If the pH of the buffer is less than 5.5, the target protein itself precipitates when heated, and if the pH exceeds 7.0, the protein derived from the host can be obtained by heating. Since the contaminants cannot be sufficiently separated and removed, the pH of the buffer should be between pH 5.5 and pH 5.5.
It must be within the range of 7.0.

The concentration at which the protein-containing substance is suspended in the buffer is not particularly limited, and the effect of the present invention can be obtained at any concentration.

The substance containing the protein suspended in a buffer solution having a pH in the range of 5.5 to 7.0 is heated at a temperature in the range of 55 to 85 ° C., and the resulting precipitate is separated and removed. Remove host-derived contaminants. Heating may be performed by, for example, bathing a buffer in which a protein-containing substance is suspended.

If the heating temperature is lower than 55 ° C., it is not possible to sufficiently separate and remove host-derived contaminants, and if the heating temperature is higher than 85 ° C. 55 ° C ~ 8
It is necessary to heat within 5 ° C.

It is more preferable that the heating temperature is in the range of 60 ° C. to 80 ° C., because the host-derived contaminants can be more sufficiently removed.

The heating time is not particularly limited, and generally, heating for 5 to 10 minutes or more is sufficient.

The precipitate generated by this heating is
The supernatant is separated and removed by centrifugation, filtration or a combination thereof. Host-derived contaminants are present in this precipitate, and the target protein is present in the supernatant.

By the above operation, a protein from which contaminants derived from the host have been removed can be obtained.

If it is necessary to further increase the purity of the protein to be obtained, the precipitate formed by heating is separated and removed, and the supernatant is further subjected to a hydroxyapatite chromatography for treatment.

As the carrier used for the hydroxyapatite chromatography, a commonly used hydroxyapatite carrier may be used. For example, commercially available ceramic hydroxyapatite (manufactured by Bio-Rad) or hydroxyapatite (manufactured by Bio-Rad) may be used.
(Manufactured by Seikagaku Corporation) or the like may be used.

In this hydroxyapatite chromatography treatment, a precipitate formed by heating is separated,
The supernatant obtained by the removal is dialyzed against a phosphate buffer having an ionic strength of 0.1 M or less, and then dialyzed with 0.1 M
By passing through a column filled with a hydroxyapatite carrier that has been equilibrated in advance with a phosphate buffer having the following ionic strength and the like, and collecting the eluted flow-through fraction, a protein with higher purity can be obtained. Obtainable.

[0049]

【Example】

[Example 1] (Removal of contaminants contained in a substance containing an HBc antigen expressed in a transformant) Escherichia coli having an HBc antigen gene sequence incorporated therein by the procedure shown in Table 1 below. (Transformants) by removing contaminants derived from the host Escherichia coli from the HBc antigen-containing substance expressed in
A highly pure HBc antigen was prepared.

[0051]

[Table 1]

(1) Transformation and Culture First, E. coli BL21 (DE3) was transformed with the expression vector pGHBc containing the entire gene sequence of the HBc antigen by the method of "D. Ha".
nahan, J .; Mol. Biol. , 166, 55
7, 1983 ". The transformed Escherichia coli (transformant) was cultured in an LB medium at 37 ° C. overnight, and then centrifuged to collect the cells.

(2) Ultrasonic crushing This was suspended in 50 mM Tris (hydroxymethyl) aminomethane buffer (pH 7.5) containing 10 mM EDTA and 1 mM PMSF, and then suspended in “Power”
Ultrasonic crushing was performed using a “Sonic Model 150” sonicator (manufactured by Yamato Kagaku), and the supernatant was recovered after centrifugation to obtain a substance containing the HBc antigen.

(3) Separation of ammonium sulfate Further, DNase I and magnesium chloride were added to the substance containing the HBc antigen, and the mixture was allowed to stand at 4 ° C. for 30 minutes. Then, ammonium sulfate (ammonium sulfate) was added so as to be 30% saturated. The mixture was stirred at 4 ° C. overnight, and then centrifuged to collect a precipitate.

(4) Heat treatment The precipitate, which is a substance containing the HBc antigen, is
The suspension was suspended in Mbis-Tris buffer (pH 6.2), and the substance containing the HBc antigen in this buffer was heated at 65 ° C for 30 minutes. Next, this was centrifuged to separate and remove the precipitate, and the supernatant was obtained as an HBc antigen-containing solution from which contaminants derived from the host (E. coli) had been removed.

(5) Hydroxyapatite chromatography treatment This HBc antigen-containing solution was dialyzed against a phosphate buffer (pH 6.8) having an ionic strength of 10 mM, and the resulting solution was previously equilibrated with the same buffer. Column packed with apatite (Bio-Rad) (diameter 6c)
m, length 6 cm).
The eluted solution was collected in 4 ml increments, and the passed-through fractions (fraction numbers 4 to 10) were collected to obtain a more pure HBc antigen.

The elution curve in this hydroxyapatite chromatography treatment is shown in FIG. In this figure, the abscissa represents the number of the fraction obtained when 4 ml of the eluted solution was collected, and the ordinate represents the absorbance at 280 nm representing the protein mass.

[0058]

FIG.

(6) Results Table 2 shows the total protein content and the recovery in each step of obtaining the HBc antigen. The recovery rate was determined by measuring the amount of HBc antigen in the sonicated supernatant obtained in the above (2) by an ELISA method, and expressed as a relative ratio when this was taken as 100%.

The measurement of the amount of HBc antigen by the ELISA method was performed as follows. To each well of a 96-well microplate (manufactured by Nunc), 50 μl of an anti-HBc antigen polyclonal antibody (manufactured by Dako) diluted 5,000-fold with phosphate buffered saline (PBS) was dispensed.
Leave at 7 ° C. overnight.

Thereafter, this solution was removed, and 200 μl of 50 mM tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.0) containing 0.5% casein was dispensed and left at 37 ° C. for 3 hours.

After removing this solution, H
50 μl of a solution containing Bc antigen was added, and the mixture was added at 37 ° C. for 3 hours.
After reacting for hours, 0.05% Tween 20 (Twee
n20) and washed 5 times with PBS.

Then, 50 mM containing 0.5% casein
Mouse anti-H diluted 5,000-fold with Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.0)
50μ of Bc antigen monoclonal antibody (manufactured by Nagase & Co., Ltd.)
The reaction mixture was reacted at 37 ° C. for 2 hours.

Further, PB containing 0.05% Tween 20
After washing 5 times with S, P diluted 500 times with PBS
OD-labeled anti-mouse IgG antibody (manufactured by Dako) was added in an amount of 50 μl each, reacted at 37 ° C. for 1 hour, and washed five times with PBS containing 0.05% Tween 20.

Next, an o-phenylenediamine solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well in an amount of 100 μl to develop color, and then the reaction was stopped by adding 50 μl of 4N sulfuric acid. Then, the main wavelength of the solution in each well is 490 nm,
The absorbance at a subwavelength of 550 nm was measured.

The amount of HBc antigen in the HBc antigen-containing solution in each step was determined from the measured absorbance using a calibration curve prepared by measuring an HBc antigen solution of known concentration in the same manner.

[0067]

[Table 2]

The presence of contaminants in each of the above steps was confirmed by SDS electrophoresis. This is shown in FIG.

In this figure, one lane is a molecular weight marker (from the top, 202,000 daltons, 13 lanes).
3,000 daltons, 71,000 daltons, 41,8
00 dalton, 30,600 dalton, 17,800 dalton, and 6,900 dalton markers), lane 2 was the sonicated supernatant fraction, lane 3 was the fractionated sediment fraction of ammonium sulfate, and lane 4 Indicates the supernatant fraction of the heat treatment, and lane 5 indicates the electrophoresis of the fraction passing through the hydroxyapatite chromatography.

[0070]

FIG. 2

From these tables and figures, it is possible to remove contaminants by the heat treatment step of heating the substance containing the HBc antigen of the present invention to separate and remove the precipitate formed.
And it was confirmed that a highly pure HBc antigen could be obtained. It was also confirmed that the hydroxyapatite chromatography treatment following the heat treatment step could highly enhance the purity of the HBc antigen.

Example 2 (Removal of Contaminants by Various Heating Temperatures) Heat treatment was carried out at various temperatures to confirm the degree of removal of host-derived contaminants to obtain highly pure HBc antigen. Was.

(1) Heat treatment The precipitate containing the HBc antigen obtained in (3) of Example 1 was washed with a 20 mM bis-Tris buffer solution (pH
6.2), and this was dispensed into 11 portions of 5 ml each. For nine substances containing HBc antigen in these buffers, the respective substances were 20 ° C., 30 ° C., 40 ° C., 5
Heat at 0 ° C, 60 ° C, 70 ° C, 80 ° C, 90 ° C or 100 ° C for 30 minutes.

Further, one of the substances containing the HBc antigen in the eleventh buffer was not subjected to the heat treatment. Next, each of them was centrifuged to separate and remove the precipitate, and the supernatant was obtained as an HBc antigen-containing solution.

(2) Hydroxyapatite chromatography treatment One of the substances containing HBc antigen in the eleventh buffer solution of the above (1) was heated at 65 ° C. for 30 minutes and then centrifuged to separate the precipitate. The supernatant was further subjected to hydroxyapatite chromatography. This hydroxyapatite chromatography treatment
The supernatant after centrifugation was dialyzed against a 10 mM phosphate buffer (pH 6.2) having an ionic strength of 10 mM, and these were filled with ceramic hydroxyapatite (manufactured by Bio-Rad) pre-equilibrated with the same buffer. Column (diameter 6cm,
6 cm in length) and flow the same buffer at a flow rate of 2.0 m.
This was carried out by flowing the solution at 1 / min and eluating a 4.0 ml aliquot, and collecting the passed-through fractions.

(3) Confirmation of the presence of HBc antigen and contaminants by SDS electrophoresis The 10 HBc antigen-containing solutions of (1) and (2)
Was subjected to SDS electrophoresis to confirm the presence of the HBc antigen and contaminants. The result is shown in FIG.

In this figure, one lane is a molecular weight marker (from the top, 94,000 dalton, 67,0
00 dalton, 43,000 dalton, 30,000 dalton, 20,000 dalton, and 14,000 dalton), and lane 2 shows the fraction of the HBc antigen-containing solution which was not subjected to the heat treatment in the above (1). Lane 3 shows the fraction of the HBc antigen-containing solution heated at 20 ° C.
The lane shows the fraction of the HBc antigen-containing solution heated at 30 ° C., the lane 5 shows the fraction of the HBc antigen-containing solution heated at 40 ° C., and the lane 6 shows the fraction of the HBc antigen-containing solution heated at 50 ° C. The lane 7 was HBc heated at 60 ° C.
Lane 8 shows the fraction of the HBc antigen-containing solution heated at 70 ° C., Lane 9 shows the fraction of the HBc antigen-containing solution heated at 80 ° C., and Lane 10 shows the fraction of the HBc antigen-containing solution.
The fraction of the HBc antigen-containing solution heated at 0 ° C., the lane 11 was the fraction of the HBc antigen-containing solution heated at 100 ° C., and the lane 12 was heated at 65 ° C. and then subjected to hydroxyapatite chromatography. HBc
Each fraction of the antigen-containing solution was electrophoresed.

[0078]

FIG. 3

From this figure, it can be seen that when the temperature at which the protein-containing substance is heated in the buffer is 50 ° C. or lower, a large number of bands are present, and the host-derived contaminants cannot be sufficiently separated and removed. It turns out that at 90 ° C. or higher, the band of the target HBc antigen disappears and the HBc antigen itself is denatured. And
When the heating temperature is in the range of 60 ° C. to 80 ° C., it can be seen that the host-derived contaminants can be sufficiently removed without adversely affecting the HBc antigen itself.

[Reference Example] (Measurement of anti-HBc antibody in serum sample) H obtained in Example 1
Measurement of anti-HBc antibody in serum sample using Bc antigen
Performed by the LISA method.

The HBc antigen obtained in Example 1 was
g / ml phosphate buffered saline (PBS)
Was dispensed into each well of a 96-well microplate (manufactured by Nunc) in an amount of 50 μl, left at 37 ° C. overnight, and then removed. Then, a tris (hydroxymethyl) aminomethane buffer containing 5% skim milk (pH
8.0) was added to each well in an amount of 200 μl, left at 4 ° C. for 3 hours, and then removed.

HBc, a commercially available anti-HBc antibody assay reagent
42 serum samples (positive samples) determined to have anti-HBc antibody by antibody rear kit (manufactured by Dynabot) [RIA method] and 42 serum samples (negative sample) determined to have no anti-HBc antibody Was diluted 100 times, and 50 μl of each was added to the above-mentioned wells and reacted at 4 ° C. for 2 hours. Thereafter, the plate was washed five times with PBS containing 0.05% Tween 20.

Next, a peroxidase-labeled anti-human IgG antibody (manufactured by Amersham) diluted 10,000 times with a tris (hydroxymethyl) aminomethane buffer solution (pH 8.0) containing 0.5% casein was used. 50 μl per well
The reaction was continued at 4 ° C. for 1 hour.

This was added to PB containing 0.05% Tween 20
After washing 5 times with S, 100 μl of o-phenylenediamine solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well, and reacted at 37 ° C. for 30 minutes.

Thereafter, 50 μl of a 4N sulfuric acid solution was added to each well to stop the reaction, and the main wavelength was 490 nm.
The anti-HBc antibody in the sample was measured by measuring the absorbance at a subwavelength of 550 nm for each well.

FIG. 4 shows the measurement results of the positive specimen, and FIG. 5 shows the measurement results of the negative specimen.

In these figures, the horizontal axis represents the specimen number of each specimen, and the vertical axis represents the absorbance at a main wavelength of 490 nm and an auxiliary wavelength of 550 nm. In the measurement of the anti-HBc antibody in this sample, it is determined that the anti-HBc antibody is present (positive) when the measured absorbance is 0.2 or more, and the antibody is detected when the measured absorbance is less than 0.2. HBc
It was determined that no antibody was present (negative).

[0088]

FIG. 4

[0089]

FIG. 5

From these figures, it can be seen that HBc obtained by the present invention
It can be seen that the results of the measurement of the anti-HBc antibody using the antigen and the results of the measurement with the commercially available anti-HBc antibody measurement reagent are completely the same in both the positive and negative samples. Therefore, it was confirmed that when the anti-HBc antibody in the sample was measured using the HBc antigen obtained according to the present invention, the anti-HBc antibody in the sample could be accurately measured.

[0091]

Industrial Applicability The method for removing contaminants contained in a substance containing a protein expressed in a transformant of the present invention can be carried out in a host-derived manner without going through very complicated and multi-step processes. This is a useful method that can separate and remove the contaminant from the protein-containing substance to obtain a high-purity protein in a large amount, simply and at low cost.

Further, the protein of the present invention can be produced in a large amount without complicated labor-intensive and multi-step processes.
It is a highly pure protein that can be obtained simply and at low cost.

[Brief description of the drawings]

FIG. 1 is a graph showing an elution curve in a hydroxyapatite chromatography treatment.

FIG. 2 is a photograph confirming the presence of HBc antigen and contaminants in each step by SDS electrophoresis.

FIG. 3 is a photograph in which the presence of HBc antigen and contaminants when heat treatment was performed at various temperatures was confirmed by SDS electrophoresis.

FIG. 4 is a graph showing the measurement results of positive samples in the measurement of anti-HBc antibodies in serum samples.

FIG. 5 is a graph showing the results of measurement of a negative sample in the measurement of an anti-HBc antibody in a serum sample.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12N 15/09 C12N 15/00 A (72) Inventor Katsuro Koike 1-37-1 Kamiikebukuro, Toshima-ku, Tokyo Foundation Cancer Research Institute Cancer Research Institute

Claims (18)

[Claims] Claims: 1. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
55 ° C. in a buffer within the range of 5.5 to pH 7.0
A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating at a temperature in the range of -85 ° C and separating and removing a precipitate formed thereby. 2. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
60 ° C. in a buffer within the range of 5.5 to pH 7.0
A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating the mixture within a temperature range of -80 ° C and separating and removing a precipitate formed thereby. 3. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
55 ° C. in a buffer within the range of 6.0 to pH 6.5.
A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating at a temperature in the range of -85 ° C and separating and removing a precipitate formed thereby. 4. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
60 ° C. in a buffer within the range of 6.0 to pH 6.5.
A method for removing contaminants contained in a substance containing a protein expressed in a transformant, comprising heating the mixture within a temperature range of -80 ° C and separating and removing a precipitate formed thereby. 5. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
55 ° C. in a buffer within the range of 5.5 to pH 7.0
Heating in the range of ８５85 ° C., separating and removing the precipitate formed by this, and then performing hydroxyapatite chromatography, comprising the contaminants contained in the substance containing the protein expressed in the transformant Removal method. 6. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
60 ° C. in a buffer within the range of 5.5 to pH 7.0
Heating at a temperature in the range of ８０80 ° C., separating and removing the resulting precipitate, and then performing hydroxyapatite chromatography, comprising the contaminants contained in the substance containing the protein expressed in the transformant Removal method. 7. A protein-containing substance is obtained by subjecting a transformant obtained by a genetic recombination technique to ultrasonic crushing,
It is obtained through one or more steps selected from enzyme treatment, surfactant treatment and ammonium sulfate fractionation treatment,
The method for removing contaminants according to any one of claims 1 to 6. 8. A hepatitis B virus core antigen-containing substance expressed in a transformant obtained by a genetic recombination technique is treated at 55 ° C. in a buffer having a pH in the range of 5.5 to 7.0. A method for removing contaminants contained in a substance containing a core antigen of hepatitis B virus expressed in a transformant, comprising heating the mixture within a temperature range of -85 ° C and separating and removing a precipitate formed thereby. . 9. A method for preparing a substance containing a core antigen of hepatitis B virus expressed in a transformant obtained by a genetic recombination technique at a temperature of 60 ° C. in a buffer having a pH in the range of 5.5 to 7.0. A method for removing contaminants contained in a substance containing a core antigen of hepatitis B virus expressed in a transformant, comprising heating at a temperature in the range of ~ 80 ° C and separating and removing a precipitate formed thereby. . 10. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
H55 in a buffer within the range of pH 5.5 to pH 7.0.
A protein from which contaminants have been removed by heating at a temperature in the range of from 0 ° C to 85 ° C, and separating and removing the resulting precipitate. 11. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
H 60 in a buffer within the range of pH 5.5 to pH 7.0.
A protein from which contaminants have been removed by heating at a temperature in the range of from 80 ° C to 80 ° C, and separating and removing the resulting precipitate. 12. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
H in a buffer within the range of 6.0 to pH 6.5.
A protein from which contaminants have been removed by heating at a temperature in the range of from 0 ° C to 85 ° C, and separating and removing the resulting precipitate. 13. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
H 60 in a buffer in the range of 6.0 to pH 6.5.
A protein from which contaminants have been removed by heating at a temperature in the range of from 80 ° C to 80 ° C, and separating and removing the resulting precipitate. 14. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
H55 in a buffer within the range of pH 5.5 to pH 7.0.
A protein from which contaminants have been removed by heating within the temperature range of from 85 ° C. to 85 ° C., separating and removing the precipitate formed by this, and then performing a hydroxyapatite chromatography treatment. 15. A substance containing a protein expressed in a transformant obtained by a genetic recombination technique,
H 60 in a buffer within the range of pH 5.5 to pH 7.0.
A protein from which contaminants have been removed by heating at a temperature in the range of from 0 to 80 ° C, separating and removing the resulting precipitate, and then performing a hydroxyapatite chromatography treatment. 16. A protein-containing substance is obtained by subjecting a transformant obtained by a genetic recombination technique to one or more steps selected from sonication, enzyme treatment, surfactant treatment and ammonium sulfate fractionation. The protein according to any one of claims 10 to 15, wherein the protein is obtained through the process. 17. A hepatitis B virus core antigen-containing substance expressed in a transformant obtained by a genetic recombination technique is added at 55 ° C. in a buffer having a pH in the range of 5.5 to 7.0. A core antigen of hepatitis B virus from which contaminants have been removed by heating in the range of 〜85 ° C. and separating and removing a precipitate formed thereby. 18. A substance containing a core antigen of hepatitis B virus expressed in a transformant obtained by a genetic recombination technique is treated at 60 ° C. in a buffer solution having a pH in the range of 5.5 to 7.0. A core antigen of hepatitis B virus from which contaminants have been removed by heating in the range of 80 ° C. and separating and removing a precipitate formed thereby.