JP3673852B2 - Protein purification method - Google Patents

Description

【００５１】
表１から判るように、各種試薬を含んだ状態であってもSephadex G-100に対するDBDの吸着率はほとんど低下していない。それどころか、0.1%Tween20を除く各種試薬を上清Aに含ませた場合、Sephadex G100に対するDBDの吸着率は向上している。このことから、デキストランビーズに融合タンパク質を吸着させるに際して緩衝液Aに各種試薬が存在した場合、融合タンパク質の吸着率を向上させることができ、より多量の融合タンパク質を精製することができることが判った。
【００５２】
８．混合時間と精製効率との関係
上清Aに含まれる融合タンパク質をデキストランビーズに吸着させるに際して、上清Aにデキストランビーズを混合した後のインキュベーション時間と融合タンパク質の回収率との関係を検討した。具体的には、デキストランビーズとしてSephadex G-100を使用し、インキュベーション時間を0〜15時間に変化させ、それぞれの場合で回収した融合タンパク質量を測定した。上清Aに含まれる融合タンパク質の全量に対する回収できた融合タンパク質の量を百分率で算出した。結果を図４に示す。
【００５３】
この図４から判るように、インキュベーション時間が長くなるのに応じて、融合タンパク質の回収率が向上している。また、インキュベーション時間を十分に設定することによって、融合タンパク質の70%以上を回収できることが判った。この結果から、上述した方法によれば、宿主細胞で発現した融合タンパク質を高収率で回収できることが明らかとなった。
【００５４】
９．融合タンパク質の溶出に用いるデキストランの検討１
デキストランビーズに吸着した融合タンパク質を溶出させる際に使用するデキストランの濃度について検討した。具体的には、融合タンパク質を吸着させたSephadex G-200を含む緩衝液Aに添加する分子量18000のデキストラン（SIGMA社製）の濃度を変化させ、それぞれの濃度で溶出させた融合タンパク質量を測定した。結果を図５に示す。
【００５５】
この図５から判るように、分子量18000のデキストランの濃度を高くするほど、融合タンパク質をより多量に溶出させることができる。特に、デキストランの濃度を20mg/ml以上とした場合には、融合タンパク質をほぼ最大に溶出することができることが判った。
【００５６】
１０．融合タンパク質の溶出に用いるデキストランの検討２
デキストランビーズに吸着した融合タンパク質を溶出させる際に使用するデキストランの分子量について検討した。具体的には、融合タンパク質を吸着させたSephadex G-100を含む緩衝液Aに、様々な分子量のデキストランを添加し、デキストランの分子量と融合タンパク質の回収率との関係を検討した。このとき、デキストランの分子量は、1KDa（Fluka社製）、5KDa（Fluka社製）、10KDa（アマシャムファルマシアバイオテク社製）、18.1KDa（SIGMA社製）、70KDa（Fluka社製）、150KDa（Fluka社製）、500KDa（アマシャムファルマシアバイオテク社製）、2000KDa（アマシャムファルマシアバイオテク社製）、5000KDa（polyscience社製）のものを用いた。結果を図６に示す。
【００５７】
図６から判るように、分子量が５〜７０ｋＤａのデキストランを使用した場合には、融合タンパク質の回収率が高い値を示している。この結果から、Sephadex G-100から融合タンパク質を効率よく精製するには、分子量５〜７０ｋＤａのデキストランを使用することが好ましいことが判った。
【００５８】
【発明の効果】
以上、詳細に説明したように、本発明によれば、デキストラン結合ドメインを付加タグとしてを用いることによって、溶出剤として生理活性物質を用いることなく、目的とするタンパク質を迅速に精製することが可能なタンパク質の精製方法を提供できる。
【００５９】
【配列表】

【図面の簡単な説明】
【図１】実施例で作製した発現ベクターの構築を説明するための図である。
【図２】上清A（レーン１）、溶出したGFP-DBD融合タンパク質を含む上清（レーン２）及び赤色蛍光-DBD融合タンパク質（レーン３）のSDS-PAGE写真である。
【図３】Sephadex G-200に結合したタンパク質量とSephadex G-200の量との関係を示す特性図である。
【図４】インキュベーション時間と融合タンパク質の回収率との関係を示す特性図である。
【図５】デキストランの濃度と溶出されたタンパク質量との関係を示す特性図である。
【図６】デキストランの分子量と融合タンパク質の回収率との関係を示す特性図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a protein purification method capable of easily isolating a target protein.
[0002]
[Prior art]
Obtaining the proteins to be studied easily and efficiently is not only very important in molecular biology, biochemistry, pharmacy and medicine, but also important in various industries such as biochemistry and pharmaceutical industries. That is. In the past, techniques for isolating / purifying the protein of interest from biological materials such as biological tissues, organs and cultured cells have been the mainstream. In this case, handling of the biomaterial is troublesome, and since the target protein is present in a small amount, it is difficult to obtain the target protein in high purity and in large quantities. In addition, even when a large amount of the target protein exists, other proteins with similar physicochemical properties to the target protein often coexist, so that only the target protein can be easily isolated / It was difficult to purify.
[0003]
On the other hand, in recent years, it has become possible to obtain a large amount of a target protein as a recombinant protein using a large-scale expression system using a recombinant gene and a host cell. In order to obtain a recombinant protein, first, a gene encoding the target protein is cloned into an expression vector. The host cell is then transformed with the expression vector. Then, by expressing a gene cloned into an expression vector in the host cell, a large amount of the target protein can be produced in the host cell.
[0004]
In addition, when a protein is purified using this mass expression system, a method using a so-called addition tag is known. That is, a large amount of a fusion protein obtained by fusing a target protein and an additional tag is expressed in a host cell. Thereafter, the lysate of the host cell is passed through a column containing an adsorbent, and an additional tag in the fusion protein is bound to the adsorbent to separate and purify the fusion protein.
[0005]
Here, polyhistidine, glutathione transferase, maltose binding domain, and the like are used as the additional tag. However, when the compatibility between these additional tags and the target protein is poor, the fusion protein does not express or forms an inclusion body even if expressed. In such a case, these problems may be solved by changing the host cell strain, examining the culture conditions, or changing the type of the additional tag. In particular, by changing the type of the additional tag, the fusion protein can be surely expressed, and formation of inclusion bodies can often be avoided. Therefore, when purifying the target protein, it is preferable to prepare a wide variety of additional tags.
[0006]
In addition, when the addition tag as described above is used, after the fusion protein is bound to the adsorbent, the adsorbent and the fusion protein are separated by acting an eluent corresponding to the type of the additional tag. As the eluent, physiologically active substances such as imidazole and glutathione are used. Therefore, the separated and purified fusion protein is present in a solution containing these physiologically active substances, and must be used after the physiologically active substance is removed by dialysis or the like. However, dialysis or the like for the purpose of removing a physiologically active substance takes a very long time, and there is a possibility that the activity of the target protein may be deteriorated through a process such as dialysis.
[0007]
[Problems to be solved by the invention]
Therefore, the present invention has been devised in view of the actual situation as described above, and by using a completely new addition tag, a target protein can be rapidly purified without using a physiologically active substance as an eluent. It is an object of the present invention to provide a protein purification method that can be used.
[0008]
[Means for Solving the Problems]
The method for purifying a protein according to the present invention that has achieved the above-described object includes a protein molecule to be purified, a dextran binding domain represented by SEQ ID NO: 1, or at least one amino acid in SEQ ID NO: 1, deletion, substitution or addition An adsorbent containing a cross-linkable dextran is mixed in a solution containing a fusion protein obtained by fusing a dextran-binding domain having an amino acid sequence and having dextran-binding activity, and then removed from the solution. The adsorbent is mixed in a solution containing dextran, and the fusion protein is eluted in the solution containing the dextran.
[0009]
In the present invention, the dextran-containing solution preferably contains dextran having a molecular weight of 5 to 70 [kDa].
Furthermore, in the present invention, it is preferable to mix dextran in a solution containing dextran at a concentration of 20 mg / ml or more.
[0010]
Furthermore, in this invention, when mixing the said adsorbent in the solution containing the said fusion protein, it is preferable to make the said solution into high salt concentration.
Furthermore, in the present invention, when the adsorbent is mixed in the solution containing the fusion protein, inhibition that inhibits non-specific adsorption between the adsorbent and a non-purified protein in the solution. It is preferable to add an agent.
[0011]
Furthermore, in the present invention, it is preferable to use dextran having a molecular weight of 5000 or less as one of the inhibitors.
Furthermore, in the present invention, the fusion protein is preferably formed by interposing a linker sequence between the protein molecule to be purified and the dextran binding domain.
Furthermore, in the present invention, the fusion protein eluted in the solution containing the dextran is preferably treated with an enzyme that specifically cleaves the linker sequence.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the protein purification method according to the present invention will be described in detail.
1. Fusion protein expression
In this method, any protein can be purified. In this method, first, a fusion protein obtained by fusing a protein to be purified (hereinafter referred to as a target protein) and a domain that specifically binds to dextran, that is, a dextran binding domain (hereinafter referred to as DBD). To prepare.
[0013]
Here, DBD means one comprising the amino acid sequence shown in SEQ ID NO: 1, or one having an amino acid sequence in which at least one amino acid in SEQ ID NO: 1 is deleted, substituted or added and having dextran binding activity. To do. DBD is a domain of glucosyltransferase produced by a given oral bacterium (Cariogenetic streptococci) (Abo. H. et al., J. Bacteriol. 173: 989-996, 1991).
[0014]
In the present method, the fusion protein may be one in which a linker sequence is interposed between the target protein and the DBD. Here, examples of the linker sequence include sequences that are specifically recognized and cleaved by an enzyme such as a protein hydrolase. For example, as the linker sequence, the amino acid sequence shown in SEQ ID NO: 2, which is recognized and cleaved by thrombin, or the factor X (manufactured by SIGMA) and factor Xa (manufactured by Amersham Pharmacia Biotech) are recognized and cleaved. The amino acid sequence shown in SEQ ID NO: 3 can be exemplified.
[0015]
When preparing a fusion protein, an expression vector that expresses the fusion protein is prepared, a host cell is transformed with the expression vector, and the transformed host cell is cultured. This expression vector has a gene encoding the target protein downstream of the promoter, a base sequence encoding a linker sequence if necessary, and a gene encoding DBD. The expression vector may have a gene encoding DBD downstream of the promoter, a base sequence encoding a linker sequence if necessary, and a gene encoding the target protein in this order. An example of a gene encoding DBD is shown in SEQ ID NO: 4.
[0016]
The gene encoding the target protein can be prepared using a normal gene cloning method. Specifically, a gene encoding a target protein can be prepared using a method as described in Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989).
[0017]
In addition, a gene encoding DBD can be prepared, for example, using the method described in Abo. H. et al., J. Bacteriol. 173: 989-996, 1991. In addition, for example, a chromosyltransferase-producing chromosomal DNA library of oral bacteria (Cariogenetic streptococci) is prepared, a clone having a glucosyltransferase gene is screened from the chromosomal DNA library, and a gene encoding DBD is screened from the screened clone. Can be prepared.
[0018]
Examples of vectors that can be used when constructing an expression vector include pUC series (Takara Shuzo), pET series (Novagen), pGEX series (Amersham Pharmacia Biotech), pCAL series (STRATAGENE) PYES2 (manufactured by Invitrogen), pYEUra3 (manufactured by Clontech), pFastBac1 (manufactured by Gibco), and the like. When inserting a gene encoding the target protein and a gene encoding DBD into these vectors, a conventionally known recombinant DNA technique (see Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989)) is used as appropriate. Can be used.
[0019]
Examples of host cells that can be used include Escherichia coli, Bacillus subtilis, yeast cells, insect cells, and animal cells. These host cells are preferably selected as appropriate according to the type of expression vector. In other words, the type of vector can be selected depending on the type of host cell to be used. For example, when Escherichia coli is used as a host cell, the expression vector is preferably constructed using the pUC series, pET series, pGEX series, or pCAL series.
[0020]
As a method for transforming a host cell, any conventionally used method can be used. For example, a method of transforming by applying heat shock after making Escherichia coli competent cells, a polyethylene glycol method, an electroporation method, a particle gun method, or the like can be used.
[0021]
A large amount of the transformed host cell is cultured, and the fusion protein is expressed inside the host cell. When the lac promoter is used as the promoter in the expression vector, the gene encoding the fusion protein downstream of the lac promoter is expressed by adding isopropyl 1-thio-β-D-galactoside (IPTG) to the culture medium. be able to. Further, even when a vector having a T7 promoter (when a host cell having a DE3 gene is used) or a tac promoter is used, similar expression induction using IPTG is possible.
[0022]
2. Recovery of fusion protein
After inducing the expression of the fusion protein, the host cells are recovered, and the recovered host cells are dissolved in a buffer solution and lysed by sonication or the like. When lysing, it is not limited to ultrasonic treatment, and a conventionally known method can be used. For example, a lysozyme / DNase method and a french press method can be mentioned.
[0023]
After lysing the host cells, the solution is centrifuged and the supernatant is recovered. An adsorbent containing a crosslinkable dextran is mixed with the collected supernatant. Here, the adsorbent may have any shape / form such as a bead shape or a dendrid shape. For example, it is preferable to use a bead-shaped adsorbent for ease of handling. As beads containing a crosslinkable dextran (hereinafter referred to as dextran beads), for example, Sephadex beads (manufactured by Amersham Pharmacia Biotech) and Sephacryl beads (manufactured by Amersham Pharmacia Biotech) can be used.
[0024]
The fusion protein having DBD can be adsorbed to the dextran beads by adding the dextran beads and then leaving them to stir. At this time, it is preferable to remove contaminating proteins contained in the supernatant. For example, by increasing the salt concentration in the supernatant, coexisting proteins other than the fusion protein adsorbed on the dextran beads can be removed. Moreover, it is preferable to add the inhibitor which inhibits nonspecific adsorption | suction with a non-purification protein and dextran beads to a supernatant liquid. Examples of the inhibitor include surfactants such as polyoxyethylene-pt-octylphenyl ether and low molecular weight dextran. By allowing this inhibitor to be present in the supernatant at a predetermined concentration, it is possible to remove contaminating proteins other than the fusion protein adsorbed to the dextran beads.
[0025]
Here, since low molecular weight dextran has a relatively low affinity for DBD in the fusion protein, by adding low molecular weight dextran to the supernatant, it is possible to confuse other than the fusion protein adsorbed to the dextran beads. Protein can be removed. Here, the low molecular weight dextran means one having a molecular weight of 5000 [Da] or less. Examples of the low molecular weight dextran include dextran T1 (manufactured by Fluka).
Next, dextran beads are recovered from the supernatant by centrifugation or the like. When collecting dextran beads, not only centrifugation but also natural standing can be used.
[0026]
Next, the collected dextran beads are washed. For example, dextran beads can be washed by adding a buffer to dextran beads and stirring well. During this washing, the contaminating protein can be removed by adding a salt and / or a surfactant as described above. Further, this washing is preferably performed a plurality of times, for example, about 5 times.
[0027]
The washed dextran beads are then mixed into a solution containing dextran. As a result, the fusion protein bound to the dextran beads is separated from the dextran beads. Then, the fusion protein can be purified by removing only the dextran beads from the solution.
[0028]
Here, when Sephadex G-100 beads are used to recover the fusion protein, it is preferable to use a dextran having a molecular weight of 5 to 70 kDa. When dextran having a molecular weight of 5 to 70 kDa is used, the recovery rate of the fusion protein can be improved. In other words, when dextran having a molecular weight of less than 5 kDa or dextran having a molecular weight of more than 70 kDa is used, the fusion protein may not be separated from the dextran beads, and the recovery rate of the fusion protein may be reduced. .
[0029]
In addition, it is preferable to mix dextran in a solution containing dextran beads at a concentration of 20 mg / ml or more. By making the dextran concentration in the solution 20 mg / ml or more, the recovery rate of the fusion protein can be improved. In other words, when the dextran concentration in the solution is less than 20 mg / ml, the fusion protein may not be separated from the dextran beads, and the recovery rate of the fusion protein may be reduced.
[0030]
On the other hand, the solution containing the fusion protein contains dextran. However, since dextran is an inactive substance, it does not adversely affect the fusion protein even if it exists in a solution containing the fusion protein. Therefore, the fusion protein can be purified by the steps described above except when dextran is positively removed.
[0031]
In addition, when removing dextran, methods, such as a dialysis and a chromatography, can be used. Specifically, since DBD in the fusion protein is an acidic domain, the fusion protein can be adsorbed on an anion exchange resin, for example, DEAE-Toyopearl650M (manufactured by Tosoh Corporation), and dextran can be removed. The fusion protein adsorbed on the anion exchange resin can be eluted with a salt such as NaCl. When low molecular weight dextran (for example, molecular weight 5 KDa) is used for elution, this can be removed by dialysis.
[0032]
In addition, when a fusion protein in which a linker sequence is interposed between the target protein and DBD is purified, the target protein and DBD can be separated by treating with an enzyme that specifically cleaves the linker sequence. . For example, when the linker sequence of SEQ ID NO: 2 is interposed, the target protein and DBD can be separated by adding thrombin to the solution containing the purified fusion protein and processing. Thereafter, the DBD is recovered by dextran beads or chromatographed using properties such as the isoelectric point of the target protein, whereby the DBD can be removed and the target protein can be purified with high purity.
[0033]
Moreover, although the example which performs the purification method of the protein which concerns on this invention in batch type was given in the description mentioned above, the protein purification method which concerns on this invention refine | purifies a protein continuously so that it may demonstrate below. The so-called column type may be used.
That is, a column in which dextran beads as described above are packed is prepared. Then, after lysing host cells expressing the fusion protein, the collected supernatant is passed through this column. Thereby, the fusion protein can be adsorbed to dextran beads inside the column. Subsequently, a predetermined buffer or the like is passed through the column for the purpose of removing contaminating proteins present in the column. Next, the fusion protein adsorbed on the dextran beads can be eluted by passing a solution containing dextran through the column.
[0034]
Thus, the protein purification method according to the present invention can purify the target fusion protein even when the column formula is applied. The solution containing the fusion protein eluted from the column will contain dextran. However, since dextran is an inactive substance, it does not adversely affect the fusion protein even if it exists in a solution containing the fusion protein. Therefore, the fusion protein can be purified by elution from the column, except when dextran is positively removed.
[0035]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, the technical scope of this invention is not limited to a following example.
[Example 1]
In this example, a green fluorescent protein (hereinafter referred to as GFP) used as a model protein in various fields was used as a target protein.
[0036]
1. Construction of expression vector
As shown in FIG. 1, DBD / pUC18 (Abo, H. et al., J. Bacteriol. 173: 989-996, 1991) in which a gene encoding DBD is linked downstream of the lac promoter in pUC18 (Takara Shuzo Co., Ltd.). Prepare). In this DBD / pUC18, a gene encoding GFP was linked between the lac promoter and the gene encoding DBD (multicloning site) to construct GFP-DBD / pUC18 as shown in FIG.
[0037]
2. Transformation
As a host cell, E. coli BL21 (DE3) strain (Novagen), which is a protease-deficient strain, was used. This E. coli BL21 (DE3) strain was transformed with GFP-DBD / pUC18 by the heat shock method.
[0038]
Specifically, Escherichia coli BL21 (DE3) strain was mixed with 20 mM β-mercaptoethanol, allowed to stand in ice for 10 minutes, and then added with GFP-DBD / pUC18 and stored in ice. After storage in ice 30, heat treatment was performed at 42 ° C. for 30 seconds, and then stored in ice for 2 minutes. After adding 42 ° C SOC medium, the cells were cultured with shaking at 37 ° C for 1 hour. This bacterial cell culture was cultured overnight at 37 ° C. on an LB plate containing chloramphenicol and ampicillin.
[0039]
3. Culture of transformed cells
The transformed Escherichia coli BL21 (DE3) strain was precultured in LB liquid medium containing ampicillin at 37 ° C. with shaking for 12 to 16 hours. Subsequently, 2.5 ml of the preculture solution was added to 500 ml of TPM medium, and cultured at 37 ° C. while appropriately measuring the absorbance. When the absorbance at a wavelength of 600 nm reached 0.5, 0.5 mM IPTG was added and cultivation was continued at 20 ° C. for 3 hours. In addition, expression of the gene downstream of the lac promoter is induced by addition of IPTG.
[0040]
At this time, when the culture broth was observed visually, it showed a light green color. In addition, when the Escherichia coli BL21 (DE3) strain in the culture solution was observed using a fluorescence microscope (OLYMPUS IX50), it was confirmed that GFP-derived fluorescence was emitted over the entire cell. In E. coli BL21 (DE3) strain, no inclusion bodies were formed.
[0041]
4). Recovery of fusion protein
E. coli BL21 (DE3) strain was collected by centrifuging the culture solution at 4 ° C. and 7500 × g for 2 minutes. The recovered E. coli BL21 (DE3) strain was suspended in buffer A (composition: 10 mM Tris-acetate (pH 8.0), 1 mM Mg-acetate and 150 mM K-acetate), and then centrifuged to obtain E. coli BL21 ( The strain DE3) was collected. The collected Escherichia coli BL21 (DE3) strain had a clear yellow-green color.
[0042]
Next, the recovered E. coli BL21 (DE3) strain was suspended in buffer A containing 5 mM EDTA and 1 mM PMSF, and then subjected to sonication to lyse. Then, it was centrifuged at 4 ° C. and 10000 × g for 30 minutes. Here, it was confirmed that the supernatant fraction obtained by centrifugation (hereinafter referred to as “supernatant A”) had a bright yellow-green fluorescent color. The electrophoresis pattern of the obtained supernatant fraction is shown in lane 1 in FIG.
[0043]
Next, Sephadex G-200 was added to the supernatant A at a final concentration of 2.5%, and kept at 4 ° C. on a rotator for 30 minutes. Then, Sephadex G-200 was recovered as a precipitate fraction by centrifuging at 4 ° C. and 2000 × g for 20 seconds. Next, an excessive amount of buffer A was added to the collected Sephadex G-200 and mixed well. Thereafter, centrifugation was performed under the same conditions, and Sephadex G-200 was collected again as a precipitate fraction. By repeating this washing treatment 5 times, the non-specific adsorbed protein adsorbed to Sephadex G-200 was removed.
[0044]
Next, Sephadex G-200 after the washing treatment was suspended in buffer A, and dextran having a molecular weight of 18000 (manufactured by SIGMA) was added to 10 mg / ml and mixed well. Subsequently, Sephadex G-200 was precipitated by centrifuging at 4 ° C. and 2000 × g for 20 seconds, and a supernatant exhibiting a yellowish green fluorescent color was collected. The collected supernatant was analyzed by SDS-PAGE / CBB staining (lane 2 in FIG. 2) and densitometry, and it was found that the purity of the fusion protein exceeded 95%.
[0045]
5. Other examples
The fusion protein was purified in the same manner as described above except that the target protein was red fluorescent protein instead of GFP. When the finally collected supernatant was analyzed by SDS-PAGE / CBB staining, it was found that the fusion protein could be purified to a very high purity as shown in lane 3 in FIG.
[0046]
6). Examination of adsorption amount of fusion protein
The amount of fusion protein adsorbed on Sephadex G-200 was examined. Specifically, glucosyltransferase (GTF) and DBD described in Abo, H., et al., J. Bacteriol. 173: 989-996, (1991), respectively, were purified by a conventionally known method to obtain a predetermined concentration. Are prepared for each protein. Then, various volumes of Sephadex G-200 suspended in buffer A and protein are mixed. The mixed solution was allowed to stand for 30 minutes while being kept at 4 ° C. on a rotator, and then centrifuged at 4 ° C. and 2000 × g for 60 seconds to precipitate Sephadex G-200, and the supernatant was recovered.
[0047]
The protein concentration contained in the collected supernatant was measured using BIO-RAD PROTEIN ASSAY (manufactured by BIO-RAD). The amount of protein adsorbed on Sephadex G-200 was calculated from the difference between the protein concentration measured without mixing Sephadex G-200 and the protein concentration in the collected supernatant. The relationship between the amount of protein bound to Sephadex G-200 and the amount of Sephadex G-200 is shown in FIG.
[0048]
FIG. 3 shows that 30 μmole (4.1 mg) is adsorbed on GTF and 70 μmole (2.1 mg) is adsorbed on DBD per 1 ml of dextran beads (Sephadex G-200). Further, when the same experiment was conducted with an incubation time of 3 hours, 103 μmol (14.2 mg) of GTF and 97 μmol (2.9 mg) of DBD were adsorbed. From these results, it was proved that the fusion protein could be sufficiently adsorbed to the dextran beads by the above-described method, and that the fusion protein could be sufficiently purified.
[0049]
7). Effects of coexisting reagents
When various reagents were present in the supernatant A when the fusion protein was adsorbed on the dextran beads, it was investigated what effect the adsorption characteristics have. Specifically, Sephadex G-100 was used as dextran beads, and the adsorption rate of DBD to Sephadex G-100 was measured in the supernatant A containing various reagents. The results are shown in Table 1.
[0050]
[Table 1]

[0051]
As can be seen from Table 1, the adsorption rate of DBD on Sephadex G-100 is hardly lowered even in the state containing various reagents. On the contrary, when various reagents except 0.1% Tween20 are included in the supernatant A, the adsorption rate of DBD to Sephadex G100 is improved. From this, it was found that the adsorption rate of the fusion protein can be improved and a larger amount of the fusion protein can be purified when various reagents are present in the buffer A when the fusion protein is adsorbed to the dextran beads. .
[0052]
8). Relationship between mixing time and purification efficiency
When the fusion protein contained in the supernatant A was adsorbed to the dextran beads, the relationship between the incubation time after mixing the dextran beads with the supernatant A and the recovery rate of the fusion protein was examined. Specifically, Sephadex G-100 was used as dextran beads, the incubation time was changed from 0 to 15 hours, and the amount of fusion protein recovered in each case was measured. The amount of fusion protein recovered with respect to the total amount of fusion protein contained in supernatant A was calculated as a percentage. The results are shown in FIG.
[0053]
As can be seen from FIG. 4, the recovery rate of the fusion protein is improved as the incubation time becomes longer. It was also found that 70% or more of the fusion protein can be recovered by setting the incubation time sufficiently. From this result, it became clear that according to the above-described method, the fusion protein expressed in the host cell can be recovered in high yield.
[0054]
9. Examination of dextran used for elution of fusion protein 1
The concentration of dextran used to elute the fusion protein adsorbed on the dextran beads was examined. Specifically, the concentration of 18000 molecular weight dextran (manufactured by SIGMA) added to buffer A containing Sephadex G-200 adsorbed with the fusion protein was varied, and the amount of fusion protein eluted at each concentration was measured. did. The results are shown in FIG.
[0055]
As can be seen from FIG. 5, the higher the concentration of dextran having a molecular weight of 18000, the more the fusion protein can be eluted. In particular, it was found that when the concentration of dextran was 20 mg / ml or more, the fusion protein could be eluted almost at the maximum.
[0056]
10. Examination of dextran used for elution of fusion protein 2
The molecular weight of dextran used to elute the fusion protein adsorbed on the dextran beads was examined. Specifically, dextran having various molecular weights was added to buffer A containing Sephadex G-100 to which the fusion protein was adsorbed, and the relationship between the molecular weight of dextran and the recovery rate of the fusion protein was examined. At this time, the molecular weight of dextran is 1 KDa (Fluka), 5 KDa (Fluka), 10 KDa (Amersham Pharmacia Biotech), 18.1 KDa (SIGMA), 70 KDa (Fluka), 150 KDa (Fluka) Manufactured), 500 KDa (manufactured by Amersham Pharmacia Biotech), 2000 KDa (manufactured by Amersham Pharmacia Biotech), and 5000 KDa (manufactured by polyscience). The results are shown in FIG.
[0057]
As can be seen from FIG. 6, when dextran having a molecular weight of 5 to 70 kDa is used, the recovery rate of the fusion protein is high. From this result, it was found that it is preferable to use dextran having a molecular weight of 5 to 70 kDa in order to efficiently purify the fusion protein from Sephadex G-100.
[0058]
【The invention's effect】
As described above in detail, according to the present invention, by using a dextran binding domain as an additional tag, it is possible to rapidly purify a target protein without using a physiologically active substance as an eluent. A method for purifying various proteins.
[0059]
[Sequence Listing]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram for explaining the construction of an expression vector prepared in an example.
FIG. 2 is an SDS-PAGE photograph of supernatant A (lane 1), supernatant containing eluted GFP-DBD fusion protein (lane 2), and red fluorescence-DBD fusion protein (lane 3).
FIG. 3 is a characteristic diagram showing the relationship between the amount of protein bound to Sephadex G-200 and the amount of Sephadex G-200.
FIG. 4 is a characteristic diagram showing the relationship between the incubation time and the recovery rate of the fusion protein.
FIG. 5 is a characteristic diagram showing the relationship between the concentration of dextran and the amount of eluted protein.
FIG. 6 is a characteristic diagram showing the relationship between the molecular weight of dextran and the recovery rate of the fusion protein.

Claims (6)

A protein molecule to be purified, and a dextran-binding domain represented by SEQ ID NO: 1 or a dextran-binding domain having an dextran-binding activity having an amino acid sequence in which at least one amino acid in SEQ ID NO: 1 has been deleted, substituted or added solution in a solution containing a fusion protein fused by mixing adsorbent containing a crosslinking dextran, then dextran of the adsorbent extracted from the above-mentioned solution, molecular weight 5 ~ 70 [kDa] of A method for purifying a protein, which comprises mixing in and eluting the fusion protein in the solution.   The method for purifying a protein according to claim 1, wherein dextran is mixed in the solution containing dextran at a concentration of 20 mg / ml.   The method for purifying a protein according to claim 1, wherein when the adsorbent is mixed in a solution containing the fusion protein, the solution is made to have a high salt concentration. When the adsorbent is mixed in the solution containing the fusion protein, an inhibitor having a molecular weight of 5000 or less as an inhibitor that inhibits nonspecific adsorption between the adsorbent and a non-purified protein in the solution . Dextran is added , The protein purification method of Claim 1 characterized by the above-mentioned.   The method for purifying a protein according to claim 1, wherein the fusion protein comprises a linker sequence interposed between a protein molecule to be purified and a dextran binding domain.   The method for purifying a protein according to claim 1, wherein the fusion protein eluted in the solution containing dextran is treated with an enzyme that specifically cleaves the linker sequence.

Images