JP2000044597A - PRODUCTION OF RECOMBINANT SYPHILIS 47 kDa ANTIGEN - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for purifying the subject 47 kDa antigen through the process for eluting a GST-fused protein with a molecular weight of 47 kDa from an immobilized glutathione column by preventing the fused protein from being precipitated. SOLUTION: This method comprises producing the surface antigen of Treponema pallidum with a molecular weight of 47kDa using a technique of fusion with GST(glutathione-S-transferase) in Escherichia coli; wherein sarkosyl and/or octyl glucoside is used for preventing occurrence of precipitation pref. at 0.5-1.0 wt.% and/or about 1.0 wt.% in concentration for sarkosyl and/or octyl glucoside, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a 47 kDa syphilis antigen protein by expressing it in Escherichia coli, extracting and purifying it. For more information, syphilis 47kD
The present invention relates to a method for producing a 47 kDa antigen protein using a suitable surfactant when producing an a antigen protein by a fusion method with glutathione-S-transferase.

[0002]

2. Description of the Related Art Syphilis is a disease caused by Treponema pallidum (Treponema Pallidum, hereinafter abbreviated as Tp). Whether or not syphilis is infected is diagnosed by examining whether or not anti-Tp antibodies are present in the blood by immunoassay. Tp
A large number of surface antigens are present on the cell surface, and the above-mentioned immunoassay is carried out using an antigen-antibody reaction between this surface antigen and the anti-Tp antibody in the sample. The surface antigen of Tp used in an infectious disease test is obtained by crushing live cultured Tp bacteria by ultrasonic treatment, homogenizer treatment, or the like, further solubilizing this, and using it as an antigen.

[0003] However, culture of Tp is performed using rabbit testes, and it is difficult to obtain Tp in large quantities.

[0004] In order to solve this problem, it has been proposed to clone a gene encoding the surface antigen of Tp, mass-produce the surface antigen by genetic engineering, and use it for the above-mentioned immunoassay. The main Tp surface antigens have a molecular weight of 47 kDa, 42 kDa, 17 kDa and 15 kD
The antigen of a is known. The genes encoding these surface antigens have already been cloned and their amino acid sequences have been determined (INFECTION AND IMMUNITY, Vol. 5).
7, No.12, pp.3708-3714.1989; Molecular microbiolo
gy (1990) 4 (8), 1371-1379; INFECTION AND IMMUNITY,
Vol.61, No.4pp.1202-1210. 1993).

In general, Escherichia coli (Escherichia coli) is used to produce an antigen protein by genetic engineering. Several vectors have been reported for inducing the expression of foreign proteins in E. coli (Harris, Williamson, Genetic Ent.
gineering (1983) Vol. 4, p128-175, Academic Press L
ondon; Marston, Biochem. J., (1986) 240, p1-12;
Masokintyre et al., Int. J. Parasitol. (1987) 17, p5
A problem common to most of these systems, however, is that denaturing reagents are required to maintain protein solubility. Denaturation can be expected to alter the antigenicity of the foreign polypeptide and destroy all functional activity.

[0006] In order to solve this problem, a foreign protein is fused with the enzyme glutathione-S-transferase (parasitic worm Cystsoma japonica, hereinafter GST), preferably the carboxyl terminus of GST, and the fusion protein is expressed. A method for refining is known (Japanese Patent Publication 6
-81596). The fusion protein is generally soluble and can be purified from the soluble components (lysate) of E. coli under non-denaturing conditions, eg, by affinity chromatography on a fixed glutathione column. It is stated that this fusion protein can be used as is, since the foreign protein retains its antigenicity and functional activity. In addition, it is described that only the desired foreign protein can be purified by cleaving the binding site between GST of the fusion protein and the foreign protein with an appropriate protease.

[0007] However, when the foreign protein is highly hydrophobic such as a membrane protein, the fusion protein may be insoluble even in the method described in Japanese Patent Publication No. 6-81596, making purification difficult. Although the above publication describes that the fusion protein is solubilized and purified using a surfactant in that case, the publication described in the publication is not always sufficient, and the optimal interface for each foreign protein is individually determined. The choice of activator is necessary.

[0008]

[Problems to be Solved by the Invention]
In the method described in the publication, a series of plasmid expression vectors (pGEX) for fusing a foreign protein to the carboxyl terminus of GST via an amino acid sequence cleavable by a site-specific protease have been constructed.

The present inventors have inserted the gene for the 47 kDa antigen into a vector (for example, pGEX-2) that can be cleaved by thrombin, a type of site-specific protease.
The gene was recombined to form a GST-47 kDa fusion protein. Then, according to the method described in Japanese Patent Publication No. 6-81596, expression purification of the 47 kDa antigen was attempted as follows. First, the recombinant p containing the 47 kDa gene
Escherichia coli containing the GEX plasmid was added to Isopropylthi
By adding o-β-D-galactoside (IPTG), the fusion protein was induced and expressed in the cells.

[0010] When the cells were disrupted by sonication or the like, centrifuged, and the supernatant (lysate) was extracted and analyzed, the fusion protein was soluble and was abundant in the lysate. The lysate thus obtained was mixed with immobilized glutathione beads and washed well. As a result, only the fusion protein could be adsorbed to the immobilized glutatine. However, when an attempt was made to elute the fusion protein from the immobilized glutathione with an excess of glutathione solution (50 mM Tris-HCl solution containing 5 mM reduced glutathione), the eluted fusion protein immediately precipitated and the desired 47 kDa could not be obtained. It became clear. Probably because the fusion protein tends to precipitate at high concentrations.

[0011] Then, the present inventors took out the precipitate of the produced fusion protein and used the surfactant {1% Triton-x100, 1% Tween described in JP-B-6-81596.
n20, 10 mM dithiothreitol (DTT), 0.
An attempt was made to solubilize the fusion protein with 03% sodium dodecyl sulfate (SDS)｝, but the fusion protein was not sufficiently dissolved. However, in the case of SDS, the precipitate was dissolved at a concentration of 0.5%. Since it is industrially difficult to remove the precipitate of the fusion protein, 0.5% SD is added to the excess glutathione solution in advance.
When we tried to elute the fusion protein with S added,
Most of the fusion protein was recovered without precipitation.
However, when thrombin was allowed to act on the fusion protein recovered with 0.5% SDS to attempt cleavage, the fusion protein was hardly cleaved and the desired 47 kDa antigen was not obtained. Probably because SDS inhibits the function of thrombin.

The present invention has been made in view of the above situation, and
It is an object of the present invention to provide a method for purifying a 47 kDa antigen by preventing a fusion protein from forming a precipitate in a step of eluting a fusion protein with kDa from an immobilized glutathione column.

[0013]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that sarkosyl and / or octyl glucoside, which are surfactants, are previously added to an excess glutathione solution. As a result, the present inventors have found that the generation of a precipitate can be avoided, and the obtained 47 kDa antigen can be purified by cleaving the obtained fusion protein with thrombin and applying it to a fixed glutathione column, thereby completing the present invention.

That is, the recombinant syphilis 47 according to the present invention.
The method for producing the kDa antigen is based on the molecular weight 4 of Treponema pallidum.
A 7 kDa surface antigen was expressed in E. coli by glutathione-
This is a method characterized by using sarkosyl and / or octyl glucoside in order to prevent the generation of precipitates when preparing by a fusion method with S-transferase.

Hereinafter, the present invention will be described in detail.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A series of plasmid expression vectors (pGEX) for fusing a foreign protein to the carboxyl terminus of GST via an amino acid sequence cleavable by a site-specific protease have already been constructed ( JP-B-6-81596 or a catalog of Pharmacia. In addition, the 47 kDa gene can be inserted into a vector (for example, pGEX-2 or the like) that can be cleaved by thrombin, which is a kind of site-specific protease, and the gene can be recombined to form a fusion protein. These genetic engineering techniques are known.

Recombinant pGEX containing 47 kDa gene
Escherichia coli into which the plasmid has been introduced is described in
According to the method described in Japanese Patent Application Laid-Open Publication No. H10-86, a fusion protein can be induced and expressed by adding IPTG thereto. The cells of Escherichia coli expressing the fusion protein are disrupted by sonication or the like, and the resulting lysate is mixed with immobilized glutathione (eg, glutathione sepharose beads) and washed well. Can be adsorbed. The fusion protein is eluted from the immobilized glutathione with an excess glutathione solution (for example, a 50 mM Tris-HCl solution containing 5 mM reduced glutathione). As a result, the fusion protein elutes, but the eluted fusion protein immediately precipitates.

Thus, if sarkosyl and / or octylglucoside are added as a surfactant in advance to an excess glutathione solution, the fusion protein can be efficiently recovered. In order to efficiently recover the fusion protein, the concentration of sarkosyl should be at least 0.5%, preferably 0.5 to
The concentration of octylglucoside is preferably about 1%.

After the addition of the surfactant, the recovered fusion protein can be cleaved by thrombin without removing the surfactant. However, GST and 47k were contained in the sample of the fusion protein reacted with thrombin.
A 37 kDa fragment is generated in addition to the Da antigen.
The amount of the 37 kDa fragment increases as the concentration of thrombin increases. Since thrombin does not cleave GST, this fragment is thought to be from the 47 kDa antigen. When a 47 kDa amino acid sequence was frequently searched, a thrombin cleavage sequence (Leu Va1) was found.
Pro Arg G1y Ser ) (Pr
o Va1 Pro Arg Ile Ser ) is present at a site about 130 amino acids away from the carboxy terminus.
Cleavage of the 47 kDa antigen at this site generates a 37 kDa fragment. Therefore, the 37 kDa fragment is considered to be a partial degradation product of the 47 kDa antigen. Since the site of this similar sequence reacts slowly with thrombin, the amount can be reduced to several percent or less by optimizing the amount of thrombin. When recovered with 0.5% sarcosyl, thrombin is most preferably 0.1 unit for 0.1 mg of fusion protein, and when recovered with 1% octyl glucoside, 3 units of thrombin is used for 0.1 mg of fusion protein. .5 units is optimal. The fact that sarkosyl requires significantly less units is presumed that sarkosyl acts on the fusion protein to expose the cleavage site.

The GST after cleavage of the fusion protein and the uncleaved fusion protein can be removed together by adsorption to immobilized glutathione, and the purified 47 kDa antigen and 37 kDa
Only Da remains. Since 37 kDa is considered to be several percent or less, it is considered that there is no problem in application to a raw material of a diagnostic agent. By the above method, 2 to 5 mg of the target 47 kDa antigen can be obtained per liter of Escherichia coli culture solution.

[0021]

The present invention will be described more specifically with reference to the following examples.

Reference Example 1 Construction of Plasmid for Expression of Fusion Protein and Expression in Escherichia coli (Incorporation of 47 kDa Gene into pGEX-4T) Syphilis was purified from rabbit testis, a syphilis subculture, and genomic DNA was extracted therefrom. did. Using this as a template, a known 47 kDa DNA sequence (Weigel et al., INFECTION AND
Based on IMMUNITY (1992) Vol.60, No.4, p1568-1576)
P using primers prepared using NA synthesizer
A 47 kDa antigen DNA fragment was obtained by the CR method. This DNA fragment has a D corresponding to amino acids 20 to 434 excluding the N-terminal peptide which has been excised in the native antigen.
Contains the NA sequence. For cloning into an expression vector, both ends of this DNA fragment are restricted to Bam.
An HI recognition sequence was added. A vector pG constructed to express this DNA fragment into a fusion protein with GST
The resulting plasmid was inserted into the BamHI site of EX-4T (Pharmacia) and the resulting plasmid was named pGEX47T.
In pGEX47T, the gene sequence has been recombined so that the carboxyl terminus of GST is fused with the 47 kDa antigen via the amino acid sequence at the cleavage site of thrombin, which is a site-specific protease (see FIG. 1).

(Expression of fusion protein) An overnight culture of Escherichia coli JM109 strain into which pGEX47T was introduced was used to prepare a fresh LB medium (Bacto Trypton 10 g, Bacto Yeast extract 5 g, NaC
(1: 5 g / l), diluted 1: 100 in 1 liter, cultured at 37 ° C. for about 3 hours, and added IPTG (manufactured by Sigma) (0.5 mM). After further culturing for about 5 hours, the centrifuged cells were subjected to 30 ml of an STE buffer (10 mM Tris-HCl) containing 100 μg / ml lysozyme (Sigma).
pH 8.0, 150 mM NaCl, 1 mM EDTA). Further 1.5 ml of 1M dithiothreitol (DTT), 0.
3 ml of 100 mM PMSF and 4.5 ml of 1
After adding 0% sarkosyl, loose sonication (Bran
The bacteria were lysed on ice (1 minute at 20% power with a son microchip). 14.8 ml of 10%
Add Triton-X100, 10 minutes at 4 ° C,
Centrifugation was performed at 000 × g. The supernatant was mixed with 8 ml of 50% glutathione-Sepharose beads (Pharmacia) at room temperature in a 50 ml polypropylene tube placed on a rotating platform. Before use, put beads on P
Pre-swelled in BS buffer, washed twice with the same buffer,
Stored as a 50% (vol / vol) solution in PBS at 4 ° C. After adsorption, the beads were centrifuged for 10 seconds (500
× g) and washed three times with 50 ml of PBS.
To confirm the fusion protein, add 50 μl of beads to 100
boil in μl sample buffer, 10% SDS-PAG
E analysis was performed followed by 0.05% Coomassie blue staining. As a result, GST (molecular weight of about 27 kDa) and 47
A fusion protein of about 74 kDa fused with the kDa antigen was detected. Thus, glutathione-Sepharose beads to which the GST-47 kDa fusion protein was attached could be prepared.

Normally, elution of the fusion protein from the glutathione-sepharose beads is carried out by using an excess glutathione solution # 5.
5 mM Tris containing mM reduced glutathione (Sigma)
This is performed by adding -HCl (pH 8.0) to compete with free glutathione. In the case of the present GST-47 kDa fusion protein, it precipitates and cannot be recovered.

Example 1 Elution of Fusion Protein by Sarkosyl (Recovery of Fusion Protein from Glutathione-Sepharose Beads) The fusion protein was eluted from glutathione-Sepharose beads prepared in Reference Example 1 by using an excess glutathione solution containing sarkosyl. This was performed using Excess glutathione solution containing sarkosyl at the same concentration as the beads (1%, 0.5%, 0.1%) is added to the glutathione-Sepharose beads, and reacted for 10 minutes while rotating on a rotating platform. And centrifuged at 500 × g to obtain a supernatant containing the fusion protein. This operation was performed three times. Boil each supernatant sample in an equal volume of sample buffer,
10% SDS-PAGE analysis followed by 0.05% Coomassie blue staining.

As a result, it was found that by adding 0.5% or 1.0% sarkosyl to the excess glutathione solution, precipitation was prevented and the fusion protein was efficiently recovered (see FIG. 2). .

(Site-specific proteolysis of fusion protein by thrombin) [0027] When the recovered fusion protein is incubated with thrombin (Pharmacia), G
In addition to ST, the 47 kDa antigen, and the uncleaved fusion protein, a 37 kDa fragment was generated (see FIG. 3). The 37 kDa fragment is considered a partial degradation product of the 47 kDa antigen.

In order to determine the optimal concentration of thrombin,
Each concentration (0.9, 0.7, 0.5, 0.3, 0.1, 0.1%) was determined based on 0.1 mg of the recovered fusion protein containing 0.5% sarkosyl as it was.
0.05 units) of thrombin and added at 25 ° C for about 16
Reacted for hours. The samples were boiled in the same volume of sample buffer and subjected to 10% SDS-PAGE analysis followed by 0.05% Coomassie blue staining. As a result, the effective cleavage with the highest amount of 47 kDa and the lowest amount of 37 kDa is 0.1 m.
The ratio of enzyme to substrate was found to be 0.1 units per g of fusion protein (see FIG. 3).

(Purification of 47 kDa protein) In order to purify the 47 kDa antigen from the cleaved fusion protein, the fusion protein cleaved with thrombin was added to 10 mg / 50 mg of the fusion protein.
of 50% glutathione-Sepharose beads (Pharmacia) pre-swelled and washed, and adsorbed on a rotating platform for 10 minutes. A portion of the supernatant obtained by centrifugation at 500 xg was boiled in an equal volume of sample buffer, analyzed by 10% SDS-PAGE, then 0.05%
% Coomassie blue staining was performed to analyze the proteins in the supernatant.

As a result, GST and the uncleaved fusion protein were both removed by adsorption to glutathione-Sepharose beads, and the purified 47 kDa antigen and 37 kDa
It was confirmed that only the residue remained (see FIG. 4). 37
Since kDa is considered to be 1% or less, it does not hinder the application to a raw material or the like of a diagnostic agent.

Example 2 Elution of fusion protein with octyl glucoside (Recovery of fusion protein from glutathione-sepharose beads) Elution of the fusion protein from glutathione-sepharose beads prepared in Reference Example 1 was carried out using excess octyl glucoside. This was performed using a glutathione solution. Each concentration (2%, 1%, 0.5%, 0.1%)
%) Of octylglucoside was added to glutathione-Sepharose beads, and the mixture was reacted for 10 minutes while rotating on a rotating platform, and then centrifuged at 500 × g to obtain a supernatant containing the fusion protein. This operation was performed three times. Each supernatant sample was boiled in an equal volume of sample buffer and subjected to 10% SDS-PAGE analysis followed by 0.05% Coomassie blue staining.

As a result, it was found that the addition of 1% octyl glucoside to the excess glutathione solution prevented precipitation and efficiently recovered the fusion protein (see FIG. 5). However, the effect of preventing precipitation was not sufficiently exhibited with 2% octyl glucoside.

(Site-specific proteolysis of fusion protein by thrombin) The recovered fusion protein was incubated with thrombin (Pharmacia) to give G
In addition to ST, the 47 kDa antigen, and the uncleaved fusion protein, a 37 kDa fragment was generated (FIG. 6).
(A)). The 37 kDa fragment is considered a partial degradation product of the 47 kDa antigen.

To determine the optimal concentration of thrombin,
To each of 0.1 mg of the collected fusion protein directly containing% octyl glucoside, thrombin at each concentration (10, 7.5, 5, 3.5, 1 unit) was added and reacted at 25 ° C. for about 16 hours. 10% S by boiling in sample buffer equal to sample
DS-PAGE analysis was performed followed by 0.05% Coomassie blue staining. As a result, it was found that the effective cleavage having the largest amount of 47 kDa and the smallest 37 kDa had an enzyme-to-substrate ratio of 3.5 units to 0.1 mg of the fusion protein (see FIG. 6 (A)). In this case, the 37 kDa fragment is considered to be about 3%.

Next, in order to further reduce the content of the 37 kDa fragment, an attempt was made to examine a buffer solution during hydrolysis of the fusion protein with thrombin. HEPES containing 1 liter of 1% octyl glucoside
Buffer (20 mM HEPES pH 7.5, 150 m
M NaCl, 100 mM KCl, 2.5 mM Ca
The solution was dialyzed against Cl ₂ , 1 mM DTT and the buffer solution was changed. To examine the optimal concentration of thrombin, thrombin at each concentration (10, 7.5, 5,3.5, 1 unit) was added to 0.1 mg of the fusion protein, and reacted at 25 ° C. for about 16 hours. 10% by boiling in the same volume of sample buffer as the sample
SDS-PAGE analysis followed by 0.05% Coomassie
Blue staining was performed. The results showed that the effective cleavage with the highest amount of 47 kDa and the lowest of 37 kDa had an enzyme to substrate ratio of 5 units per 0.1 mg of fusion protein. In this case, the 37 kDa fragment is considered to be 1% or less (see FIG. 6 (B)).

(Purification of 47 kDa protein) To purify the 47 kDa antigen from the cleaved fusion protein, HEPE was used.
The fusion protein cleaved with thrombin in S buffer was
10 ml of a pre-swelled and washed 50 ml of 0 mg
% Glutathione-Sepharose beads (Pharmacia) and adsorbed on a rotating platform for 10 minutes. A portion of the supernatant obtained by centrifugation at 500 × g was boiled in an equal volume of sample buffer, and 10% SDS-PAGE was performed.
Analysis followed by 0.05% Coomassie blue staining was performed to analyze proteins in the supernatant.

As a result, the GST and the uncleaved fusion protein were both removed by adsorption to glutathione-Sepharose beads, and the purified 47 kDa antigen and 37 kDa
Only the remaining was confirmed (see FIG. 7). 37
Since kDa is considered to be 1% or less, it does not hinder the application to a raw material or the like of a diagnostic agent.

[0038]

According to the present invention, in the step of eluting a fusion protein of GST and 47 kDa from an immobilized glutathione column, the fusion protein is prevented from forming a precipitate.
The kDa antigen can be purified.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the construction of plasmid pGEX47T.

FIG. 2 is an electrophoretogram showing the results of elution of a fusion protein from glutathione-Sepharose beads using an excess glutathione solution containing sarkosyl. Excess glutathione solution containing sarkosyl at the same concentration as the beads (1%, 0.5%, 0.1%) is added to the glutathione-Sepharose beads, and reacted for 10 minutes while rotating on a rotating platform. And centrifuged at 500 × g to obtain a supernatant containing the fusion protein. This operation was performed three times (lanes 1, 2, and 3 indicate the first, second, and third times, respectively). Lane B is obtained by directly heating the beads at 100 ° C. for 3 minutes to release the fusion protein in order to analyze the remaining fusion protein that could not be recovered. laneM indicates a size marker. Each sample was boiled in an equal volume of sample buffer and subjected to 10% SDS-PAGE analysis followed by 0.05% Coomassie blue staining.

FIG. 3 is an electrophoretogram showing the results of cleaving the recovered fusion protein with thrombin. In order to examine the optimal concentration of thrombin, each concentration (lane1) was compared with 0.1 mg of the recovered fusion protein containing 0.5% sarkosyl as it was.
-6: 0.9, 0.7, 0.5, 0.3, 0.1, 0.
(0.5 units) of thrombin was added and reacted at 25 ° C. for about 16 hours. Boil in sample buffer equal to sample
0% SDS-PAGE analysis followed by 0.05% Coomassie blue staining. laneM indicates a size marker.

FIG. 4 is an electrophoretogram showing a 47 kDa antigen purified from a cleaved fusion protein. The thrombin-cleaved fusion protein was mixed with 10 ml of 50% glutathione-sepharose beads (Pharmacia) pre-swelled and washed for 50 mg, and adsorbed on a rotating platform for 10 minutes. A portion of the supernatant obtained by centrifugation at 500 × g was boiled in an equal volume of sample buffer, and 10% S
The protein in the supernatant was analyzed by DS-PAGE analysis followed by 0.05% Coomassie blue staining.

FIG. 5 is an electrophoretogram showing the results of elution of a fusion protein from glutathione-sepharose beads using an excess glutathione solution containing octylglucoside. Each concentration (2%, 1%, 0.5%,
0.1%) octyl glucoside in excess glutathione solution is added to the glutathione-Sepharose beads,
After reacting for 10 minutes while rotating on a rotating platform, the mixture was centrifuged at 500 × g to obtain a supernatant containing the fusion protein. This operation was performed three times (lanes 1, 2, and 3 indicate the first, second, and third times, respectively). Lane B is obtained by directly heating the beads at 100 ° C. for 3 minutes to release the fusion protein in order to analyze the remaining fusion protein that could not be recovered. laneM indicates a size marker. Each sample was boiled in an equal volume of sample buffer and 10% SDS-
PAGE analysis followed by 0.05% Coomassie blue staining was performed.

FIG. 6 (A) is an electrophoretogram showing the result of cleaving a fusion protein recovered with an excess glutathione solution with thrombin. In order to examine the optimal concentration of thrombin, each concentration (lane 1 to 5:10, 7.5,
5, 3.5, 1 unit) of thrombin and add about 1 at 25 ° C.
The reaction was performed for 6 hours. The samples were boiled in the same volume of sample buffer and subjected to 10% SDS-PAGE analysis followed by 0.05% Coomassie blue staining. laneM indicates a size marker. (B) An electrophoretogram showing the results of thrombin cleavage after replacing the fusion protein recovered with an excess glutathione solution with a HEPES buffer containing 1% octylglucoside. In order to examine the optimal concentration of thrombin, each concentration (lane 1 to 5:10, 7.
(5, 5, 3.5, 1 unit) thrombin was added and reacted at 25 ° C. for about 16 hours. Boil in sample buffer equal to sample and 10% SDS-PAGE analysis, then 0.05%
Coomassie blue staining was performed. laneM indicates a size marker.

FIG. 7 is an electrophoretogram showing a 47 kDa antigen purified from a cleaved fusion protein. Thrombin-cleaved fusion protein in HEPES buffer was
ml of 50% glutathione-Sepharose beads (Pharmacia) pre-swelled and washed, and adsorbed on a rotating platform for 10 minutes. 500 × g
A portion of the supernatant obtained by centrifugation at boil in an equal volume of sample buffer, 10% SDS-PAGE analysis, then 0.0%
The protein in the supernatant was analyzed by 5% Coomassie blue staining.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12P 21/02 C12R 1:19)

Claims (1)

[Claims] 1. The use of sarcosyl and / or octylglucoside to prevent the occurrence of precipitation when preparing a surface antigen having a molecular weight of 47 kDa of Treponema pallidum in E. coli by fusion with glutathione-S-transferase. Recombinant syphilis 47k characterized by
A method for producing a Da antigen.