JP3624389B2 - Method for isolating protein from spent culture after culturing BCG bacteria - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for isolating a useful protein from a protein excreted by BCG bacteria from a culture solution of BCG bacteria.
[0002]
[Prior art]
Conventionally, a method of isolating MPB64 and MPB70 from a small amount of culture solution obtained by culturing BCG bacteria for a long period of 5 to 10 weeks is isolated by Nagai et al. Using DEAE Sephadex A-50 and DEAE Sepharose. The method was published in American Society for Microbiology, Vol. 52, No. 1 p293-302, published in April 1986 by American Society for Microbiology.
A method for producing the MPB64 protein using genetic engineering is known from Japanese Patent Application Laid-Open No. 1-247094.
[0003]
However, in the former method, the starting material is a culture solution for long-term culture, and the amount is extremely small. The latter is a method in which the MPB64 gene is incorporated into the host, the host is cultured, and then MPB64 is taken out from the host, and the operation is extremely troublesome.
[0004]
[Problems to be solved by the invention]
The present invention is a method for separating useful proteins typified by MPB64, MPB70, etc. from among 300 kinds of proteins excreted from the cells during cultivation of BCG bacteria. This is to provide the market with a method for efficiently isolating these proteins, and to provide the market with a reagent for measuring delayed type hypersensitivity reactions mainly comprising the isolated protein.
[0005]
[Means for Solving the Problems]
In order to achieve the above object, this method invention uses a column having different properties from the remaining culture solution obtained by removing BCG bacteria from the culture solution after culturing BCG, loads the remaining culture solution, and uses a buffer solution. Elution and fractionation sequentially into multiple parts, select the fraction containing the protein to be isolated from these, concentrate the fraction, and dialyze it. In the method of purifying the target protein by performing the same column purification method at least twice with a buffer solution containing
As a starting material, the BCG bacterium was cultured in a culture solution for 8 days using a Tokyo strain, and the remaining culture solution (hereinafter simply referred to as a used culture solution) from which the BCG bacterium was collected was used. As the pretreatment, the used culture solution is heated to 40 to 45 ° C., and this is first filtered to remove residual BCG Tokyo bacteria, and then the protein having a molecular weight of 5000 or less and the culture solution components are removed by filtration. The spent culture solution is concentrated, then ammonium sulfate (hereinafter simply referred to as AS) is added to the concentrated solution to 60% saturation to precipitate the total protein, and the total protein is separated by centrifugation. It is set as the method of isolating protein from the used culture solution after culture | cultivation of BCG bacteria characterized by supplying to a process.
[0006]
In order to achieve the above object, a DEAE Sepharose CL-6B column is used as a column in the column purification method of the second stage of the column purification method of the method for isolating a protein from the spent culture solution. As a buffer, equilibrate with 500 ml of 30 mM Tris buffer (pH 7.5) containing 3 M (mole) urea, supply the protein after the pretreatment in the column purification method, and add 0 to 0.1 NaCl in the same buffer. The buffer containing 200 mM was supplied in order from the lowest concentration while increasing the NaCl concentration continuously and fractionated into several sections with an elution rate of 0.5 ml / min. Selected,
Then, it is preferable that the urea is separated in the process of concentrating the selected fraction protein and then dialyzing.
[0007]
In order to achieve the above-mentioned problem, this method invention
a. First stage (pretreatment process)
After culturing for 8 days in a culture solution using BCG bacteria Tokyo strain, the remaining culture solution (hereinafter simply referred to as a used culture solution) from which the BCG bacteria have been collected is used as a starting material. The mixture is heated to 45 ° C., and this is first filtered to remove residual BCG Tokyo bacteria, and then the protein having a molecular weight of 5000 or less and the culture solution components are removed by filtration. Concentrated to 350,
Next, AS is added to this concentrated solution to 60% saturation to precipitate the total protein, the protein is separated by centrifugation, and the separated protein is supplied to the next step.
b. Column purification process [0008]
Second stage The protein according to the first stage is loaded onto a phenyl sepharose CL-4B column (diameter 25 mm, volume 80 ml), 10 mM Tris-HCl (HCl) buffer containing AS, and the AS concentration in turn containing 500 mM AS. 450 to 600 ml in total is supplied from the higher AS concentration while decreasing the concentration stepwise or continuously from the one to the one not containing it at all, and the eluate is sequentially fractionated by 10 ml.
10 μl sampled from each fraction was mixed with 50 μl of Bio-Rad protein assay reagent, the absorbance was measured, the elution pattern was determined, and further, the fraction containing the protein of the desired molecular weight was selected by electrophoresis, The eluate of this fraction is concentrated and further dialyzed.
[0009]
Third stage A buffer obtained by adding 0 to 200 mM NaCl to a 30 mM Tris-HCl buffer solution using a DEAE Sepharose CL-6B column (diameter 15 mm, volume 75 ml) equilibrated with 3M urea. Supplying the solution while continuously increasing the NaCl concentration,
The eluate is fractionated at an elution rate of 0.5 ml / min.
Next, as in the second step, the absorbance is measured to obtain an elution pattern, a fraction containing the target protein is selected, and then the selected fractionated protein is concentrated to obtain a concentrated protein solution.
[0010]
Step 4 The concentrated protein solution obtained in the previous step was used for equilibration using a Sephacryl S200HR column equilibrated with 500 ml of 10 mM Tris-HCl buffer containing 10% ethylene glycol (EG) and NaCl. Elute with the same buffer and fractionate sequentially.
Next, as in the third step, the absorbance is measured to determine the elution pattern, the fraction containing the target protein is selected, and then the selected fraction protein is concentrated and dialyzed to obtain a concentrated protein solution.
[0011]
Fifth Stage The concentrated protein solution of the fourth step was prepared by equilibrating a DEAE Sepharose CL-6B column containing no urea with 500 ml of 30 mM Tris-HCl buffer (pH 8.7), and using 30 mM Tris-HCl buffer (pH 8). 7) Elution with NaCl with an elution solution whose concentration is continuously increased from 50 mM to 100 mM, and fractionate sequentially.
Next, a fraction is selected by the same method as in the previous step, concentrated and dialyzed to obtain the target protein.
A method for isolating a protein from a spent culture solution after culturing BCG, characterized by comprising the first step to the fifth step as described above.
[0012]
In order to achieve the above object, in the first to fifth methods of isolating a protein from a spent culture solution after culturing the BCG bacteria, the target protein to be isolated is MPB64. It may be a feature.
[0013]
In order to achieve the above object, in the first to fifth methods of isolating a protein from a spent culture solution after culturing the BCG bacteria, the target protein to be isolated is MPB59, MPB64. MPB70 and MPB80, and in some cases, at least two kinds of proteins are isolated in parallel during the same time period in the second to fifth stages.
[0014]
[Effects of the Invention]
In the method invention according to claim 1, as a starting material, a spent culture solution in which BCG bacteria produced when producing a BCG preparation are cultured is used. Prior to isolating the protein of interest from the various proteins excreted in the medium, the used culture broth is heated to 40 ° C. to 45 ° C., so that the viscosity of the culture broth is reduced. In the filtration, the fluidity is improved, and the remaining BCG bacteria are first removed, and then the protein having a molecular weight smaller than the molecular weight of the target protein and the culture solution components are filtered in a short time.
[0015]
In the method invention according to claim 2, in addition to the action of the invention according to claim 1, in the second column purification method, a DEAE Sepharose CL-6B column is used and 3M (mole) urea is used as a buffer. Equilibrated with 500 ml of a 30 mM Tris buffer solution (pH 7.5) containing the solution, supplied with the protein after the pretreatment in the column purification method, and a NaCl solution containing 0 to 200 mM NaCl in the same buffer solution as above. Since the concentration is supplied in order from the lower concentration while the concentration is gradually increased, impurities bound to or incorporated into the protein are bound to urea and removed. Immediately after this stage, the eluate is dialyzed to remove the urea from the eluate, thereby changing the isolated protein and causing little action to weaken the activity.
[0016]
In the invention described in claim 3, in addition to the effects of the invention described in claims 1 and 2, the target protein is purified from the used culture broth by at least one of the first to fifth steps. Acts to isolate and isolate.
In particular, when there are two or more proteins to be isolated in the second stage, a plurality of fractions are selected independently, and in each subsequent stage, purification is performed independently for each fraction. Makes possible action.
In the invention described in claim 4, in addition to the action described in claim 3, it acts to isolate MPB64 as a target protein with high purity.
[0017]
In the invention of claim 5, in addition to the action of claim 3, as the target protein, at least two of MPB59, MPB64, MPB70 and MPB80 are independently and simultaneously separated in the second stage. And then, in parallel with each fractionated protein, each of them has the effect of isolating with high purity.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Materials and methods Culture medium:
Bacteria used in Sauton synthetic culture:
Mycobacterium bovis BCG Tokyo (hereinafter simply referred to as BCG bacteria), which is a seed strain manufactured by Nippon BCG Manufacturing Co., Ltd.
The BCG bacterium was transplanted to the sorton medium and surface-cultured at 37 ° C. without aeration. Bacterial cells cultured on the 8th day were collected by filtration for vaccine production, and the used culture solution after collection was collected as a starting material.
column:
DEAE Sepharose CL-6B, Sephacryl S200HR, Sepharose CL-4B (Sweden, Uppsala, Pharmacia LKB) were used.
[0019]
Sample concentration:
Millipore Pellicon System XX42PEL60 (Bedford, Millipore, MA, USA) and Amicon YM-3 membrane (Amicon Inc., MA, USA) Using.
[0020]
Two-dimensional electrophoresis (hereinafter simply referred to as 2D-E):
As 2D-E, a Millipore investigator 2-D electrophoresis system was used.
In the first dimension, isoelectric focusing was performed at 18000 V / hr in a range of pI3 to 10 using 9.5 M urea containing a 5.5% base-acid group ampholite mixture.
The second dimension is 12.5% polyacrylamide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter simply referred to as SDS-PAGE) was performed at 16000 mW using a gel (polyacrylamide gel).
[0021]
reagent:
The one for Millipore 2-D electrophoresis system was used. The gel was stained with a silver staining reagent (Japan, Tokyo, Daiichi Chemical Co., Ltd.). For the measurement of molecular weight, a standard protein (manufactured by Biorad Laboratory, Richmond, CA, USA) was used.
As other reagents, those not particularly noted were those manufactured by Wako Pure Chemical Industries, Ltd.
[0022]
Embodiment 1
Next, an embodiment including the method invention according to claims 1 to 5 will be described.
First stage (pretreatment)
First, 80 to 100 liters of the spent culture solution as the starting material is heated to 40 ° C. to 45 ° C. and filtered with No. 2 filter paper (Tokyo, Japan, manufactured by Toyo Filter Paper Co., Ltd.). The remaining BCG cells are separated by filtration, and then supplied to the Millipore, Pellicon, cassette, and apparatus while maintaining the temperature at 40 ° C to 45 ° C.
The filter of this Millipore Pericon Cassette PLCC00005 device is used to remove small molecules with a molecular weight of 5000 or less, such as glycerin and other media components.
In this way, the initial starting material is concentrated to 300-400 ml. In order to save time, this concentration may be performed by dividing into several sections and using a plurality of the above-mentioned Millipore Pellicon cassettes PLCC00005.
[0023]
The spent culture medium as the starting material is heated to the temperature range as described above, thereby reducing the viscosity and facilitating passage through a small molecule filter having a molecular weight of 5000 or less. Helps reduce time.
The concentrate is further filtered through Millipore Membrane Millipak 60 (manufactured by 0.22 μm Millipore Co., Ltd.) to completely remove BCG cells.
[0024]
To the concentrated culture filtrate, ammonium sulfate was added to 60% saturation to precipitate the total protein. The precipitate mixture is cooled and stored in a koji or refrigerator, and then separated by centrifugation at 6000 rpm, and the supernatant is removed.
FIG. 1 shows 2D-E (two-dimensional electrophoresis) of this concentrate. Proteins secreted by BCG are scattered as many independent spots depending on the isoelectric point and molecular weight. For example, MPB64 (26 kDa) is indicated by an arrow.
[0025]
Second stage (protein affinity purification with phenyl sepharose)
The total protein precipitate obtained from the first step is loaded onto a Phenyl Sepharose CL-4B column.
This column uses 500 mM AS: 150 ml, 200 mM AS: 50 ml, 100 mM AS: 150 ml, AS: none 120 ml in order from ammonium sulfate (AS) concentration of 500 mM to not containing 10 mM Tris HCl buffer reduced in each step. Elute at room temperature.
[0026]
Thus, the eluate is fractionated by 10 ml. Samples of 10 μl are taken from each fraction, mixed with 50 μl of 5-fold diluted Bio-Rad reagent (Protein Assay Bio-Rad), and the absorbance at 600 nm is measured with a microplate reader. An example of the elution pattern is shown in FIG.
In FIG. 2, for example, the peak of MPB64 has already been announced. Therefore, when purifying MPB64, the fraction containing MPB64 is estimated by detecting MPB64 based on the elution pattern and molecular weight using SDS-PAGE. Select the fraction containing these.
In the second stage, when there is a target protein over one or more fractions, these plural fractions are selected.
[0027]
As a specific method of SDS-PAGE, a standard protein such as MPB64 and MPB70 is used at a gradient of 10 to 20% polyacrylamide gel and a multi-gel kit (manufactured by Daiichi Chemicals, Tokyo, Japan) at 40 mA. The protein is stained with a silver staining reagent, and a fraction containing a protein corresponding to the standard protein is selected.
A fraction containing the protein of interest such as MPB64 is concentrated through an Amicon YS-3 membrane.
[0028]
The concentrate is placed in a slide-A-riser cassette (Pierce, Rockford, Ill., USA) and dialyzed overnight at 4 ° C. against 10 mM Tris HCl buffer, pH 7.5.
The dialyzed material was passed through a Millipore Millex GV filter (0.22 μm), and the amount of protein was quantified by the Raleigh method.
[0029]
Third stage (Purification by DEAE-Sepharose column containing urea) DEAE Sepharose CL-6B column (diameter 15 mm, volume 75 ml) was equilibrated with 500 ml of 30 mM Tris-HCl buffer (pH 7.5) containing 3 M urea. Turned into.
The concentrate from the second stage is supplied to the DEAE Sepharose CL-6B column, and NaCl (0 to 200 mM) having a concentration gradient is added to the buffer of the same component used for the equilibration and eluted.
[0030]
Elution rate is 0.5 ml per minute at 4 ° C., and the eluate is fractionated 46 to 100 in 5 ml fractions.
The absorbance at 600 nm is measured for each of these fractions using the Bio-Rad reagent in the same manner as in the second step. The result is as shown in FIG.
In the same way as in the second step, the fraction sample is detected by SDS-PAGE to detect the target protein such as MPB64, and the corresponding fraction is selected and concentrated, dialyzed and filtered as in the second step. To obtain a concentrate. In the above dialysis process, the urea component added during elution is removed. The amount of protein is also quantified by the same method as in the third step.
[0031]
Fourth stage (Purification with Sephacryl column)
A Sephacryl S200HR column (25 mm diameter, 450 ml volume) is equilibrated with 500 ml 10 mM Tris-HCl buffer (pH 7.5) containing 10% ethylene glycol and NaCl at low temperature.
The concentrate obtained in the third step is supplied to the Sephacryl S200HR column, eluted with the same buffer used for equilibration at an elution rate of 0.5 ml per minute, and sequentially 5 to 46 ml in 5 ml portions. The BCA protein analysis reagent (Rockford, Pierce, Illinois, USA) was used in place of the Biorad reagent in the second stage, and the sample from each fraction was prepared in the same manner as in the third stage. The target protein is detected by absorbance measurement at 562 nm and SDS-PAGE, and the fraction corresponding thereto is selected (see FIG. 4).
[0032]
In this case, the gel is preferably stained with CBB and then with silver with higher sensitivity.
Fractions containing the protein of interest are collected, concentrated with Amicon YM-3 membrane, and dialyzed overnight at 4 ° C. in 10 mM Tris-HCl buffer (pH 8.7). This is further filtered through a 0.45 μm Millipore filter.
The amount of recovered protein is evaluated by the Raleigh method.
[0033]
5th stage (purification with DEAE-Sepharose column without urea)
In the final step of purification, the protein recovered in step 4 is fed to a DEAE Sepharose CL-6B column equilibrated with 30 mM Tris-HCl buffer (pH 8.7) containing 50 mM NaCl without urea. Elute with 500 ml of 30 mM Tris-HCl buffer containing NaCl at a concentration gradient of 50 to 100 mM at 4 ° C. at an elution rate of 0.5 ml per minute, and fractionate sequentially into 5 to 46 minutes (FIG. 5). reference).
[0034]
The target protein is detected by absorbance at 600 nm and SDS-PAGE in the same manner as in the third step.
Fractions containing the protein of interest are collected as in the previous step, concentrated, dialyzed in 10 mM Tris-HCl buffer (pH 8.7), and filtered through a Milliporemelex-GV filter (0.22 μm Millipore Co.). .
The amount of final recovered protein is measured by the Raleigh method.
[0035]
In the above-described embodiment, the isolation of MPB64 and MPB70 has been mainly described. However, methods for simultaneously isolating other useful proteins are also included in the inventions of claims 1 to 5.
When isolating these several types of proteins, it is a matter of course that the isolation after separating several types of proteins in the second stage is performed in parallel for each protein. That is, a plurality of proteins are purified simultaneously.
[0036]
In the third stage and the fifth stage described above, the concentration of the buffer additive used for elution has been described as having a concentration gradient, but the concentration of the additive additive in stages divided into 4 to 10 stages. A method of supplying sequentially is also included in this embodiment.
[0037]
Embodiment 2
15 mg of MPB59, MPB64, MPB70 and MPB80 purified according to Embodiment 1 are dissolved in 2 ml of phosphate buffer, and 0.5 g to 1.5 g of hydrophilic ointment and a small amount of tragacanth gum powder (0 0.02 to 0.05 g) and sealed in a flat container having an inner diameter of 8 to 11 mm.
Each of the above-mentioned components, weights and volumes are examples and are not limited.
[0038]
Embodiment 3
15 mg of MPB59, MPB64, MPB70 and MPB80 purified according to Embodiment 1 are dissolved in 2 ml of phosphate buffer, mixed with 1 ml of glycerin and / or 10% of polyethylene glycol, and attached to the skin. A so-called fin chamber in which the filter paper impregnated with the protein is applied to the skin in a patch test patch for testing an allergic reaction or in an aluminum dish in which the filter paper is placed in an aluminum dish. A product impregnated with 5 to 20 μl and sealed in a plastic container or an airtight bag.
Of the above proteins, each of MPB64 and MPB70 sealed in the same manner as described above may be used as a kit.
[0039]
【The invention's effect】
In the invention described in claim 1, since the used culture solution after culturing BCG bacteria is used as a starting material, when BCG bacteria are used effectively, industrial waste is effectively used. By heating at a temperature of from 45 ° to 45 °, the viscosity of the spent culture solution is significantly reduced, so that it becomes easier to pass through the filter paper and the processing time is shortened. High glycerol is removed, making subsequent handling easier. In addition, this level of heating does not cause deterioration of the protein titer.
[0040]
In the invention of claim 2, a DEAE Sepharose CL-6B column is used, and after equilibrating with 30 mM Tris buffer (pH 7.5) containing 3 M (mole) urea as a buffer, the same urea is contained. By gradually increasing the concentration of NaCl from 0 to 200 mM in the buffer solution, supplying impurities in order from the lowest concentration, and eluting the target protein, the impurities bound to or surrounded by the protein are removed, and the elution is performed. In the medium, the neutrality of the eluate is maintained, and the urea is removed from the purified protein by dialysis and filtration immediately thereafter, thereby minimizing the effect of irreversible denaturation of the protein on urea.
[0041]
In the method according to claim 3, in addition to the effects of the inventions according to claim 1 and claim 2, by adopting the method of the second step, the useful protein is fractionated into one or more kinds of proteins. can do.
Furthermore, it has the effect of improving the purity of each protein by the method after the third stage.
Each isolated protein is identified as the same protein with a purity comparable to that of the standard protein as compared with the standard protein.
[0042]
In addition, although not exemplified above, for purification of other mycobacterial antigens including other protective antigenic substances contained in the above-mentioned spent culture medium, tuberculin, and common antigens of mycobacteria Can also be applied.
[0043]
In the invention of claim 4, since the protein to be isolated in addition to the effects of claims 1 to 3 is MPB64, the isolated MPB64 is combined with other MPB70 to cause the pathogenesis of active tuberculosis and BCG positivity can be distinguished and judged.
[0044]
In the invention described in claim 5, in addition to the effects described in claims 1 to 4, the protein to be isolated is selected from two or more proteins of MPB59, MPB64, MPB70 and MPB80 in the second to fifth stages. Since the method is performed in parallel during the same time period, it has the effect of effectively and efficiently isolating two or more proteins from a single starting material.
[0045]
In the reagent for measuring delayed type hypersensitivity reaction as described above, that is, one of MPB59, MPB64, MPB70 and MPB80 isolated by the method of claim 5 is dissolved in a phosphate buffer and sealed. Can hang this reagent on filter paper and apply it to the skin of a specimen with a bandage, or inject the reagent into the skin and examine its reaction.
[0046]
When one of MPB59, MPB64, MPB70 and MPB80 isolated by the method of claim 5 is mixed with a phosphate buffer and a hydrophilic ointment and sealed in a container, the container is opened and the sample immediately The reaction can be examined by applying to the skin.
[0047]
The composition as described above, ie, 15 mg of MPB59, MPB64, MPB70 and MPB80 isolated by the method of claim 5 is dissolved at a rate of 2 ml of phosphate buffer, 1 g of hydrophilic ointment and tragacanth gum When the mixture of the powder of 0.04 g is impregnated into the patch test adhesive bandage, the reaction can be easily examined by immediately applying it to the skin of the specimen.
[0048]
A filter paper impregnated with one of MPB59, MPB64, MPB70 and MPB80 isolated by the method of claim 5 is sealed in a plastic container, a kind of airtight bag, and immediately opened. The reaction can be examined by applying to the skin of the specimen.
[0049]
Since the isolated MPB64 and MPB70 are used as separate reagents, each of which is sealed and composed of a combination of these two kinds of reagents, the presence or absence of active tuberculosis or bacterial infection can be obtained by using these in combination. Can be diagnosed.
[0050]
Experimental example 1
In the final fifth stage, 5 mg of isolated MPB64 was obtained using 100 liters of culture broth immediately after culturing BCG bacteria for 8 days by the method of Embodiment 1.
Thus, when the obtained MPB64 and MPB70 were subjected to SDS-PAGE and tested by immunoblotting using anti-MPB64 and anti-MPB70 antibodies, they showed the same reaction as standard MPB64 and MPB70, respectively.
[0051]
Experimental example 2
In addition, MPB64 and MPB70 were isolated according to Embodiment 1, and these two proteins were combined, and guinea pigs infected with M. tuberculosis and diseased and guinea pigs inoculated with BCG were prepared and tested. Guinea pigs positively converted to tuberculin by inoculation with BCG showed a positive reaction to MPB64 and MPB70, and those with active tuberculosis were positive for MPB64 but negative for MPB70.
From these results, it was easy to distinguish between active tuberculosis and positive BCG.
Experimental example 3
MPB59 has the same effect as the tuberculin reaction, and MPB80 can be used effectively for determining whether there is an allergy including this.
[Brief description of the drawings]
FIG. 1 is a distribution diagram of isoelectric points and molecular weights of various proteins by 2D-E (two-dimensional electric perturbation).
FIG. 2 is a graph showing the absorbance distribution according to the amount of protein in each fraction in the second stage and the collected fraction determined by SDS-PAGE.
FIG. 3 is a graph showing the absorbance distribution according to the protein amount of each fraction in the third stage and the collected fraction determined by SDS-PAGE.
FIG. 4 is a graph showing the absorbance distribution according to the amount of protein in each fraction in the fourth stage and the collected fraction determined by SDS-PAGE.
FIG. 5 is a graph showing the absorbance distribution according to the protein amount of each fraction in the fifth stage and the collected fraction determined by SDS-PAGE.

Claims (5)

The remaining culture solution obtained by removing BCG bacteria from the culture solution after culturing BCG bacteria is loaded with the remaining culture solution, eluted with a buffer solution, and sequentially fractionated into a plurality of parts. Select the fraction containing the protein to be isolated from the medium, concentrate the fraction and then dialyze the column purification method using the same column purification method with buffers containing different components using different properties of the column. In a method of purifying the target protein at least twice,
As a starting material, the BCG bacterium was cultured in a culture solution for 8 days using a Tokyo strain, and the remaining culture solution (hereinafter simply referred to as a used culture solution) from which the BCG bacterium was collected was used. As the pretreatment, the used culture solution is heated to 40 to 45 ° C., and this is first filtered to remove residual BCG Tokyo bacteria, and then the protein having a molecular weight of 5000 or less and the culture solution components are removed by filtration. The spent culture solution is concentrated, then ammonium sulfate (hereinafter simply referred to as AS) is added to the concentrated solution to 60% saturation to precipitate the total protein, and the total protein is separated by centrifugation. A method for isolating a protein from a spent culture solution after culturing BCG bacteria, characterized by being supplied to a process. In the column purification method in the second stage of the column purification method, a DEAE Sepharose CL-6B column is used as the column, and the buffer is 500 ml of 30 mM Tris buffer (pH 7.5) containing 3M (mol) urea. Equilibrate and supply the protein after the pre-treatment of the column purification method, and a buffer solution in which NaCl is added in an amount of 0 to 200 mM to the same buffer solution as described above, while increasing the NaCl concentration continuously and gradually from the lower concentration. Sequentially supply, fractionate into several sections with an elution rate of 0.5 ml / min, select the fraction containing the protein of interest,
The protein is isolated from the spent culture solution after culturing BCG according to claim 1, wherein the urea is separated in the process of concentrating the selected fractional protein and then dialysis. Method. a. First stage (pretreatment process)
After culturing for 8 days in a culture solution using BCG bacteria Tokyo strain, the remaining culture solution (hereinafter simply referred to as a used culture solution) from which the BCG bacteria have been collected is used as a starting material. The mixture is heated to 45 ° C., and this is first filtered to remove residual BCG Tokyo bacteria, and then the protein having a molecular weight of 5000 or less and the culture solution components are removed by filtration. Concentrated to 350,
Next, AS is added to this concentrated solution to 60% saturation to precipitate the total protein, the protein is separated by centrifugation, and the separated protein is supplied to the next step.
b. Column purification step Second stage The protein from the first stage is loaded on a phenyl sepharose CL-4B column (diameter 25 mm, volume 80 ml), 10 mM Tris-HCl (HCl) buffer containing AS, and the AS concentration in order of 500 mM. While reducing the AS concentration stepwise or continuously from one containing AS to one containing nothing at all, 450 to 600 ml of the total amount is supplied, and the eluate is fractionated in order of 10 ml.
10 μl sampled from each fraction was mixed with 50 μl of Bio-Rad protein assay reagent, the absorbance was measured, the elution pattern was determined, and further, the fraction containing the protein of the desired molecular weight was selected by electrophoresis, The eluate of this fraction is concentrated and further dialyzed.
Third stage A buffer obtained by adding 0 to 200 mM NaCl to a 30 mM Tris-HCl buffer solution using a DEAE Sepharose CL-6B column (diameter 15 mm, volume 75 ml) equilibrated with 3M urea. The solution is supplied while continuously increasing the NaCl concentration, and the eluate is fractionated at an elution rate of 0.5 ml / min.
Next, as in the second step, the absorbance is measured to determine the elution pattern, the fraction containing the target protein is selected, and then the selected fractionated protein is concentrated and dialyzed to obtain a concentrated protein solution.
Step 4 The concentrated protein solution obtained in the previous step was used for equilibration using a Sephacryl S200HR column equilibrated with 500 ml of 10 mM Tris-HCl buffer containing 10% ethylene glycol (EG) and NaCl. Elute with the same buffer and fractionate sequentially.
Next, as in the third step, the absorbance is measured to determine the elution pattern, the fraction containing the target protein is selected, and then the selected fraction protein is concentrated and dialyzed to obtain a concentrated protein solution.
Fifth Stage The concentrated protein solution of the fourth step was prepared by equilibrating a DEAE Sepharose CL-6B column containing no urea with 500 ml of 30 mM Tris-HCl buffer (pH 8.7), and using 30 mM Tris-HCl buffer (pH 8). 7) Elution with NaCl with an elution solution whose concentration is continuously increased from 50 mM to 100 mM, and fractionate sequentially.
Next, a fraction is selected by the same method as in the previous step, concentrated and dialyzed to obtain the target protein.
A method for isolating a protein from a spent culture solution after culturing BCG, characterized by comprising the first to fifth steps. 4. The method for isolating a protein from a spent culture solution after BCG culture according to the invention of claim 3, wherein the target protein is MPB64. The target protein is MPB59, MPB64, MPB70 and MPB80, and among these, two or more proteins are isolated in parallel during the same time period in the second to fifth stages. 4. The method for isolating a protein from a spent culture solution after BCG culture according to claim 3, wherein the protein is isolated from the spent culture solution after BCG culture.

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