JPH09291A - Method for synthesizing active protein in cell-free protein synthesis system - Google Patents

Abstract

(57) [Summary] [Objective] To provide a method for synthesizing an active protein in a cell-free system. [Structure] The reducing agent is removed from the synthetic reaction solution by dialysis treatment, salting-out treatment, or a combination thereof, and an oxidizing condition is applied so that the target protein retains a correct structure and the specific activity is increased.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell-free protein synthesis system, and more particularly to a method for synthesizing an active protein having a correct disulfide bond when synthesizing a protein having a disulfide bond in a protein molecule.

[0002]

2. Description of the Related Art Conventionally, a cell-free protein synthesis method has been proposed mainly as a means for mass production using a continuous system or a means for gene analysis using a batch method (Japanese Patent Publication No. 5-505095). Special table flat 6-503477
issue). By these methods, it became possible to efficiently obtain the target protein.

However, in the cell-free system, there was a problem that many of them were inactive or low in activity depending on the kind of the synthesized protein.

[0004]

In order to solve the above-mentioned problems, the form of the protein synthesized by the cell-free system is determined, and the human nerve growth factor (hNGF) having three disulfide bonds. ) Was used. As a result of analyzing the synthesized hNGF by SDS-PAGE, it was found that six cysteines in the hNGF protein molecule did not form a disulfide bond and were in a free state.

This is because in the cell-free protein synthesis system, a reducing agent such as DTT (dithiothreitol) is added as a reaction reagent, and the reaction system is in a reduced state by these, so that a disulfide bond is formed. I thought it was impossible.

[0006] Proteins in such a state tend to aggregate to form insoluble granules, and the formation of an incorrect disulfide bond causes a decrease in specific activity. In other words, it is necessary to enable a string-like protein synthesized by ribosome, which is a protein synthesizer of cells, to form an active higher-order structure. For this purpose, it is necessary to remove the reducing agent artificially added to the cell-free protein synthesis system after the protein synthesis and remove it, and keep the protein solution under oxidizing conditions in order to form a correct disulfide bond.

The present invention provides a method for increasing the specific activity by removing the reducing agent from the reaction solution containing the target protein after the synthesis reaction and subjecting it to oxidizing conditions.

[0008]

[Means for Solving the Problems] The molecular weight of the reducing agent contained in the protein synthesis reaction solution is, for example, DTT of 154, which is a low-molecular substance and can be removed by dialysis treatment or salting-out treatment. Is.

The dialysis treatment is an operation of removing a low molecular weight substance from a high molecular weight substance solution such as a protein using a semipermeable membrane, and a cellophane tube, collodion membrane or the like can be used. It is also possible to remove low molecular weight substances by ultrafiltration.

Also in the examples of the present invention, the dialysis treatment uses a cellophane tube, the cell-free protein synthesis reaction solution is put in the cellophane tube, and the dialysis treatment is carried out using a buffer solution in the range of pH 1-10 as the external dialysis solution. To do. Buffer is usually 2
A 0 mM phosphate buffer solution (pH 8.0) is used, but it is not particularly limited thereto. The temperature during the dialysis treatment may be 4 to 60 ° C., preferably 25 ° C., and the dialysis time varies depending on the type of the target protein and the type of cell-free protein synthesis reaction solution, but it is preferably 2 to 120 hours, preferably 72 hours is good. As the reducing agent is removed during this dialysis, the protein solution gradually becomes in an oxidized state and a disulfide bond is formed.

The salting-out treatment is carried out by adding a large amount of salts to a polymer electrolyte solution such as protein to reduce the solubility.
This is an operation to precipitate the target protein.
The protein can be separated and the reducing agent can be removed. As salts, phosphates and sulfates can be used,
Ammonium sulfate is preferable. The final concentration of ammonium sulfate used is in the range of 20-80%, but it is necessary to select an appropriate final concentration depending on the properties of the protein of interest.

After precipitation and separation of the protein by salting out, it is necessary to dissolve the precipitated protein in a buffer solution and then to remove salts used for salting out by dialysis. The dialysis treatment at this time can be performed in the same manner as the above dialysis treatment, but the dialysis time can be shortened. The formation of disulfide bonds is gradually performed during protein precipitation by salting out and during dialysis treatment for salt removal.

After the cell-free protein synthesis reaction, the reducing agent is removed by dialysis treatment or salting-out treatment, so that the protein solution becomes in an oxidized state and a disulfide bond is formed. , More efficient reducing agent removal and oxidation reaction are possible.
Further, the salting-out treatment is advantageous in that the target protein can be specifically precipitated and separated, so that the protein is purified.

In the case of a combination in which dialysis treatment is carried out after salting-out treatment, protein oxidation by dialysis is the main component, but as will be described later, there is a great advantage in reducing the time required for treatment.

The present invention relates to a cell-free protein synthesis system,
It is applicable to both batch type and continuous type. In the continuous method, efficient protein synthesis becomes possible by using continuous ultrafiltration.

[0016]

In the present invention, the reducing agent can be removed from the reaction solution after the protein synthesis reaction in the cell-free system by a combination of dialysis treatment or salting-out treatment followed by dialysis treatment, and the oxidizing condition can be obtained. Can retain the correct structure and increase its specific activity.

[0017]

EXAMPLE As a cell-free protein synthesis system, a transcription-translation coupling system using S-30 extract of Escherichia coli developed by Zubay et al. Was used to synthesize hNGF. The amount of hNGF contained in the reaction solution after synthesis, the dialysis treatment, and the reaction solution after ammonium sulfate salting-out treatment was converted from the amount of color developed by Western blotting using an anti-human antibody. Specific activity was also measured by neurite outgrowth in a cultured cell line isolated from chicken embryo dorsal root ganglia. A commercially available human nerve growth factor (Cosmo Bio) was used as a standard.

(1) Preparation of Escherichia coli S-30 Extract S-30 was obtained from Escherichia coli JM109 (DE3) into which the T7 polymerase gene had been inserted by the method of Zubay et al.
An extract was prepared.

(2) Plasmid DNA pT7MNGF, which is a plasmid DNA in which the hNGF gene is incorporated downstream of the T7 promoter for highly efficient transcription, was used as a template.

(3) Synthetic reaction The cell-free protein synthetic reaction was carried out at a volume of 100 μl, 37 ° C., and 2
Went in time. As a reaction solution, 16.8 μl of S-3
0 extract, 24 μg plasmid DNA, 17 μg t
RNA, 56.4 mM Tris-acetate buffer (pH
8.4) 2.75 mM DTT, 1.22 mM AT
P, 0.75 mM CTP / GTP / UTP, 27.0
mM Phosphoenolpyruvate, 0.64 mM cA
Comprised of MP, 34.6 μg folinic acid, 1.9% polyethylene glycol 6000, 11.7 mM magnesium acetate, 36 mM ammonium acetate, 72 mM potassium acetate, and 20 amino acids that make up 0.2 mM protein. The mixture was used.

(4) Dialysis After the synthesis reaction, the reaction solution was immediately cooled with ice and then transferred to a dialysis cellophane tube. Then, a dialysis treatment was carried out at 25 ° C. for 72 hours against a 1000-fold volume of 20 mM phosphate buffer (pH 8.0) to obtain hNGF protein. . The total amount of the external dialysate was replaced every 12 hours.

(5) Salting Out with Ammonium Sulfate Immediately after the synthesis reaction, the reaction solution was cooled with ice to give a final concentration of 40%.
Ammonium sulphate for 12 hours at 4 ° C
At rest. The reaction solution salted out was 15,000 rpm, 4
The precipitate was collected by centrifugation at 10 ° C for 10 minutes and resuspended in 20 mM phosphate buffer (pH 8.0).

(6) Dialysis after salting out with ammonium sulfate Immediately after the synthesis reaction, the reaction solution was cooled with ice to give a final concentration of 40%.
Ammonium sulphate for 12 hours at 4 ° C
At rest. The reaction solution salted out was 15,000 rpm, 4
The precipitate was collected by centrifugation at 10 ° C for 10 minutes and resuspended in 20 mM phosphate buffer (pH 8.0). 15,000r
It was centrifuged at pm, 4 ° C. for 10 minutes, and the supernatant was collected. The recovered supernatant was dialyzed against 1000 mM volume of 20 mM phosphate buffer (pH 8.0) at 25 ° C. for 12 hours to obtain hNGF protein.

(7) Biological activity The biological activity of hNGF synthesized by the cell-free system was measured using a primary culture system of neurons derived from dorsal root ganglia of the 8-day-old chicken. A standard preparation, a fixed amount of reaction solution, a reaction solution after dialysis treatment, and a reaction solution after ammonium sulfate salting-out treatment were administered to nerve cells, and the number of cells having nerve fiber protrusions was counted 72 hours after administration. Then, the amount of active hNGF in each reaction solution was calculated from the corresponding standard concentration. At this time, the total amount of hNGF contained in the administered reaction solution was calculated from the amount of color developed by Western blotting using an anti-hNGF antibody. HNGF having activity and total amount of hNGF in reaction solution
From the ratio of the amounts, the effects of removing the reducing agent and advancing the oxidation reaction by each of the untreated, ammonium sulfate salting-out treatment, dialysis treatment, and treatment after performing dialysis treatment after ammonium sulfate salting-out treatment are shown in comparison with FIG. As a result, it was found that the specific activity of the untreated one or the one subjected to the dialysis treatment after the dialysis treatment or the ammonium sulfate salting-out treatment was markedly improved as compared with the untreated one. As can be seen from these data, when the dialysis treatment is performed after the ammonium sulfate salting-out treatment, there is an advantage that the dialysis treatment is comparable in specific activity and the treatment time is 1/3.

[0025]

INDUSTRIAL APPLICABILITY According to the present invention, after the protein synthesis reaction, the reaction solution containing the target protein is subjected to dialysis treatment, salting-out treatment, or a combination thereof to remove the reducing agent from the reaction solution and proceed with the oxidation reaction. By doing so, the target protein can retain the correct structure and the specific activity can be increased.

[Brief description of drawings]

FIG. 1 shows the specific activity of hNGF synthesized by the cell-free protein synthesis system of the present invention.

Claims (6)

[Claims] 1. A method for synthesizing an active protein in a cell-free protein synthesis system, which comprises removing a reducing agent from a synthesis reaction solution of the cell-free protein synthesis system and applying oxidizing conditions. 2. The method for synthesizing an active protein in a cell-free protein synthesis system according to claim 1, wherein the method for removing the reducing agent is dialysis. 3. The method for synthesizing an active protein in a cell-free protein synthesis system according to claim 1, wherein the method for removing the reducing agent is a dialysis treatment after a salting-out treatment. 4. A method for synthesizing an active protein in a cell-free protein synthesis system, which comprises removing a reducing agent from a reaction solution after a protein synthesis reaction in a cell-free protein synthesis system and applying oxidizing conditions. 5. The method for synthesizing an active protein in a cell-free protein synthesis system according to claim 4, wherein the method for removing the reducing agent is dialysis treatment. 6. The method for synthesizing an active protein in a cell-free protein synthesis system according to claim 4, wherein the method of removing the reducing agent is a dialysis treatment after a salting-out treatment.