JP4265301B2 - Cell-free protein synthesizer and synthesis method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cell-free protein synthesizer, and more particularly to a cell-free protein synthesizer that has a high filtration efficiency of a component that can pass through a semipermeable membrane and is compact. The present invention also relates to a cell-free protein synthesis method using the protein synthesizer. In the present invention, the protein includes a polypeptide.
[0002]
[Prior art]
As a protein synthesis method, a cell-free protein synthesis system is known. The cell-free protein synthesis system does not require cell culture as in the case of using living cells, and is excellent in convenience. In addition, when living cells are used, there are many proteins that are difficult to express because living cells tend to exclude foreign proteins in order to maintain their own cell functions. It is difficult to be subject to the restrictions of a living body maintenance mechanism, and is excellent from this viewpoint.
[0003]
However, the cell-free protein synthesis system has the disadvantage that the reaction time is as short as about 1 to 2 hours, and the amount of synthesis is lower than that when live cells are used. For this reason, the cell-free protein synthesis system based on the batch method could not be used for mass protein synthesis.
[0004]
Aspirin et al. Put a cell-free reaction solution in an ultrafiltration device and continuously supply a solution containing substrate amino acids, ATP and GTP to the reaction solution using a pump. By removing the reaction product containing the synthesized protein from the reaction solution, cell-free protein synthesis was sustained for over 10 hours, and the amount of protein synthesis exceeded 20 times that of the conventional method (ASSpirin et al., Science, 242). 1162-1164 (1988), Japanese Patent Publication No. 7-110236). However, according to this flow method, the apparatus becomes large and the supply amount of amino acids increases, which is disadvantageous in terms of cost.
[0005]
Yokoyama et al. Put a concentrated E. coli extract, a reaction solution containing substrate amino acids, ATP, GTP, etc. inside a test tube containing a substrate solution containing substrate amino acids necessary for protein synthesis, ATP, GTP, etc. Disposable dialyzer Dispo / Dialyzer (Spectrum) was placed and incubated for continuous cell-free protein synthesis. Cell-free protein synthesis continued for 6 hours or more, and 3 mg or more of the target protein CAT was obtained per 1 ml of the reaction solution. When the substrate solution was replaced with a new one 6 hours after the start of the reaction, the synthesis reaction further continued, and finally 6 mg of the target protein was obtained (Kigawa, T. et al., FEBS Lett., 442, 15- 19, 1999). According to this dialysis method, not only ATP and GTP energy sources but also substrate amino acids are additionally supplied to the reaction solution during the synthesis. The target protein CAT is a relatively small molecule.
[0006]
Also, a so-called multi-layer method in which a reaction vessel containing a cell-free protein synthesis reaction solution is provided via a semipermeable membrane on a supply vessel containing a substrate solution containing substrate amino acids necessary for protein synthesis, ATP, GTP, etc. Is also known. For example, rapid translation system RTS500 (Roche Tyano Sticks Co., Ltd.) and Proteios (PROTEIOS, manufactured by Toyobo Co., Ltd.) can be mentioned.
[0007]
In the dialysis method and the multi-layer method, since not only ATP and GTP energy sources but also substrate amino acids are additionally supplied to the synthesis reaction solution through the semipermeable membrane, the reaction time is long. It is not preferable to retain the produced target protein for a long time in the reaction system because of degradation of protein activity and the like.
[0008]
In addition, in filtration using a filtration membrane such as an ultrafiltration membrane or a semipermeable membrane, the concentration polarization of the synthesis reaction solution that is the liquid to be treated, that is, the filtration membrane in the synthesis reaction solution, when filtration is performed. There is a problem that the concentration polarization of the component that does not pass through the filter gradually increases as the concentration is closer to the filtration membrane, and the filtration ability gradually decreases.
[0009]
[Non-Patent Document 1]
A.S.Spirin et al., Science, 242, 1162-1164 (1988)
[Patent Document 1]
Japanese Patent Publication No.7-110236
[Non-Patent Document 2]
Kigawa, T. et al., FEBS Lett., 442, 15-19, 1999
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a compact cell-free protein synthesizer with high filtration efficiency of components that can pass through a semipermeable membrane. Another object of the present invention is to provide a cell-free protein synthesis method using the protein synthesizer.
[0011]
[Means for Solving the Problems]
The present invention includes the following inventions.
(1) a first reaction tank containing a cell-free protein synthesis reaction liquid, a second reaction tank containing the synthesis reaction liquid communicated with the first reaction tank via a liquid flow path, and a first reaction A synthetic reaction tank comprising a semipermeable membrane provided in at least one of the tank, the liquid flow path and the second reaction tank;
  Means for moving and / or swinging the synthesis reaction liquid placed in the first reaction tank and the second reaction tank through the liquid flow path;,
A means for recovering the filtrate that has passed through the semipermeable membrane by filtration, and a discharge port for the filtrate recovered by the means for recovering, provided in contact with the semipermeable membrane;A cell-free protein synthesizer.
[0012]
  According to this cell-free protein synthesizer, the synthesis reaction tank includes the first reaction tank and the second reaction tank communicated with each other via the liquid flow path, and the first reaction tank and the second reaction tank. Since the means for moving and / or swinging the synthesis reaction liquid placed in the reaction tank through the liquid flow path is provided, the movement of the synthesis reaction liquid between the first and second reaction tanks is provided. And the concentration polarization of the synthesis reaction solution is suppressed by the oscillation. Therefore, filtration of components that can pass through the semipermeable membrane can be performed with high efficiency.
  Furthermore, according to this synthesis apparatus, the liquid that has passed through the semipermeable membrane can be reliably recovered.
[0015]
(2The liquid flow path is a substantially horizontal flow path that extends horizontally from the deepest part of the first reaction tank to the deepest part of the second reaction tank.)The cell-free protein synthesizer as described.
[0016]
According to this synthesizer, the synthetic reaction liquid put in the first reaction tank and the second reaction tank can be moved and / or oscillated with each other by the substantially horizontal flow passage. The substantially horizontal flow path may be formed linearly in a wide shape, or may be formed by meandering along a substantially horizontal plane.
[0017]
(3The semipermeable membrane is disposed so as to define a bottom surface of at least one of the first reaction tank, the substantially horizontal flow path, and the second reaction tank.2) Cell-free protein synthesizer.
[0018]
According to this synthesizer, filtration is performed by the semipermeable membrane disposed on the bottom surface of at least one of the first reaction tank, the substantially horizontal flow path, and the second reaction tank. The semipermeable membrane may be disposed only on the bottom surface of the substantially horizontal flow passage. In this case, the greater the area of the substantially horizontal flow passage and the larger the area of the semipermeable membrane, the higher the filtration efficiency. Moreover, the semipermeable membrane may be disposed on all bottom surfaces of the first reaction tank, the substantially horizontal flow path, and the second reaction tank. In this case, the area of the semipermeable membrane becomes larger and the filtration efficiency becomes higher.
[0019]
(4The liquid flow path is a meandering flow path that extends from the deepest part of the first reaction tank to the deepest part of the second reaction tank while meandering along a substantially vertical plane.)The cell-free protein synthesizer as described.
[0020]
According to this synthesizer, the liquids to be treated placed in the first reaction tank and the second reaction tank can be moved and / or oscillated with each other by the meandering flow passage.
[0021]
(5A semipermeable membrane is arranged to define at least one of the vertical sides of the serpentine flow passage;4) Cell-free protein synthesizer.
[0022]
According to this synthesizer, the filtration is performed by the semipermeable membrane disposed on at least one of the two vertical side surfaces of the meandering flow passage. The larger the area of the vertical side surface of the meandering passage and the larger the area of the semipermeable membrane, the higher the filtration efficiency. When the semipermeable membrane is disposed on both vertical sides of the meandering flow passage, the area of the semipermeable membrane is increased, and the efficiency of filtration is further increased.
[0023]
(6The semipermeable membrane is provided so as to be exchangeable, (1) to (5The cell-free protein synthesizer according to any one of the above.
[0024]
According to this synthesizer, the semipermeable membrane can be replaced with a new one after the completion of a predetermined cell-free protein synthesis operation, and there is no need for a washing operation of the semipermeable membrane.
[0025]
(7The semipermeable membrane is selected from the group consisting of a dialysis membrane, a semipermeable membrane, an ultrafiltration membrane, an ultrafiltration barrier, and a membrane that can capture a target substance chemically or biologically, (1) to (6The cell-free protein synthesizer according to any one of the above.
[0026]
Since the semipermeable membranes have different pore sizes, they may be selected according to the type of target protein.
[0027]
(8) The first reaction tank and / or the second reaction tank is capable of optically measuring absorption, fluorescence or scattered light of the synthetic reaction liquid, and / or the amount of the synthetic reaction liquid is optical. (1)-(7The cell-free protein synthesizer according to any one of the above.
[0028]
In this synthesizer, if the absorption, fluorescence, or scattered light of the synthesis reaction solution can be measured optically, the filtration of the synthesis reaction solution proceeds directly without collecting the sample solution during the synthesis and filtration operation. The degree and degree of purification can be measured. Further, in this synthesis apparatus, if the amount of the synthesis reaction solution can be optically confirmed, the degree of progress of filtration directly during the synthesis and filtration operation, and between the first and second reaction vessels, The state of movement and swinging can be observed.
[0029]
In the present invention, the synthetic reaction tank including the semipermeable membrane is also preferably disposable (disposable) including the semipermeable membrane. If the synthesis reaction tank is disposable, it is not necessary to wash the synthesis reaction tank including the semipermeable membrane, which is suitable for cell-free protein synthesis.
[0030]
(9The moving and / or swinging means of the synthetic reaction solution is a device that pressurizes the inside of at least one of the first reaction tank and the second reaction tank.8The cell-free protein synthesizer according to any one of the above.
[0031]
According to this synthesis apparatus, by pressurizing the inside of one of the first reaction tank and the second reaction tank, the synthesis reaction solution in the one reaction tank is reacted with the other reaction via the liquid flow path. It can be pushed into the tank, and the synthesis reaction liquid in the other reaction tank can be pulled back into the one reaction tank through the liquid flow path by stopping the pressurization and returning it to near atmospheric pressure. At the same time, the synthesis reaction solution shakes. In this way, the synthesis reaction solution is moved and swung between the first and second reaction vessels. Moreover, the filtration speed / efficiency is increased by performing filtration in a pressurized state. Movement and swinging are controlled by the degree of pressurization.
[0032]
In the present invention, a syringe pump provided with a plunger is used as a device for pressurization. The upper part of the reaction tank to be pressurized is sealed, and the space in the reaction tank is used as a sealed system, and air is sent from the syringe pump into the reaction tank and pressurized. Subsequently, when the plunger of the syringe pump is reciprocated, the synthetic reaction solution is continuously moved and swung between the first and second reaction vessels.
[0033]
(10The moving and / or oscillating means of the synthetic reaction solution has a syringe pump having two pressure ports and a plunger, a first pipe communicating from the one pressure port to the first reaction tank, An upper seal cap of the first reaction tank that penetrates the first pipe, a second pipe that leads from the other pressure port to the second reaction tank, and an upper seal of the second reaction tank that penetrates the second pipe (1) to (1) mainly composed of a cap for use and a three-way valve interposed in the middle of the second pipe9The cell-free protein synthesizer according to any one of the above.
[0034]
(11The moving and / or swinging means of the synthetic reaction solution has a syringe pump having a single pressurizing port and a plunger, a pipe leading from the pressurizing port to the first reaction tank, and penetrating the pipe. The upper seal cap of the first reaction tank and the upper seal cap of the second reaction tank are mainly composed of (1) to (9The cell-free protein synthesizer according to any one of the above.
[0035]
(12(1) to (11) Using the cell-free protein synthesizer according to any one of
Put the cell-free protein synthesis reaction solution in the first reaction tank and the second reaction tank, perform the protein synthesis reaction,
A component that can pass through the semipermeable membrane is filtered by moving the synthesis reaction liquid placed in the first reaction tank and the second reaction tank to each other and / or swinging through the liquid flow path. A cell-free protein synthesis method for removing from a synthesis reaction system.
[0036]
This cell-free protein synthesis method is a synthesis method using the above-described cell-free protein synthesizer, and the concentration of the synthesis reaction solution is determined by movement or swinging of the synthesis reaction solution between the first and second reaction vessels. Polarization is suppressed. Therefore, filtration of components that can pass through the semipermeable membrane can be performed with high efficiency.
[0037]
(13) Filtration of components that can pass through the semipermeable membrane in a pressurized state, (12) Cell-free protein synthesis method.
[0038]
According to this synthesis method, the filtration speed and efficiency are increased by performing filtration in a pressurized state. Movement and swinging are controlled by the degree of pressurization.
[0039]
(14) Components that can pass through the semipermeable membrane include degradation products formed during synthesis, (12Or (13) Cell-free protein synthesis method.
[0040]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described with reference to the drawings.
The cell-free protein synthesizer of the present invention includes a synthesis reaction tank including a first reaction tank and a second reaction tank that are communicated with each other via a liquid flow path, and a first reaction tank and a second reaction tank. And a means for moving and / or rocking the cell-free protein synthesis reaction liquid placed in the liquid crystal via the liquid flow path.
[0041]
First, the synthesis reaction tank in the cell-free protein synthesizer of the present invention will be described. FIG. 1 is a perspective view of an example of a synthesis reaction tank. FIG. 2 is a vertical sectional view of the synthesis reaction tank of FIG. FIG. 3 is a perspective view of the filtrate collecting bottom plate. In the following description, the left and right refer to FIG. 2 as a reference, the left is referred to as left and the right is referred to as right, and the front and rear refers to the front side of the page of FIG.
[0042]
Referring to FIGS. 1 and 2, the synthesis reaction tank 10 includes a first reaction tank 11 into which a cell-free protein synthesis reaction liquid L is placed, and a synthesis reaction communicated with the first reaction tank 11 via the liquid flow path 13. A second reaction tank 12 for containing the liquid L, a semipermeable membrane M, and means 31 for recovering the filtered liquid F that has passed through the semipermeable membrane M are provided.
[0043]
Both the first reaction tank 11 and the second reaction tank 12 are of a cylindrical structure having a rectangular cross section with an upper opening formed of an optically transparent plastic. The deepest portion of the right side wall 11a of the first reaction tank 11 has an opening 11b over the entire region in the front-rear direction. The deepest part of the left side wall 12a of the second reaction tank 12 has an opening 12b over the entire region in the front-rear direction. A horizontal flow passage 13 is formed in a straight line so that the opening 11b of the first reaction tank 11 and the opening 12b of the second reaction tank 12 communicate with each other. The width in the front-rear direction of the horizontal flow passage 13 is the same as the width of the opening 11b and the opening 12b so that the area of the bottom surface of the horizontal flow passage 13 is as large as possible. The front and rear side walls and the top wall of the horizontal flow passage 13 are formed integrally with the first reaction tank 11 and the second reaction tank 12.
[0044]
A semipermeable membrane M is disposed over the entire bottom surface of the first reaction tank 11, the second reaction tank 12 and the horizontal flow passage 13. That is, the bottom surfaces of the first reaction tank 11, the second reaction tank 12, and the horizontal flow passage 13 are defined and formed by the semipermeable membrane M. The semipermeable membrane M is supported and disposed on the recovery bottom plate 31 of the filtrate F and can be exchanged.
[0045]
As shown in FIG. 3, the recovery bottom plate 31 has a rectangular shape in plan view, flanges 32 and 33 (left and right flanges 32 and front and rear flanges 33) are formed on the peripheral edge, and a number of convex shapes extending in the front and rear direction on the upper surface. 34 are formed in parallel to each other and maintain a constant distance C from the front and rear flanges 33,33. Further, a discharge port 35 for the filtrate F is formed in the center portion of the bottom plate 31, and the interval C is a filtrate flow channel formed so as to become lower toward the center portion in the left-right direction. The filtrate F is collected. The semipermeable membrane M is supported by a large number of convex shapes 34.
[0046]
FIG. 4 shows a modification of the means for collecting the filtrate F that has passed through the semipermeable membrane M. About the 1st reaction tank 11, the 2nd reaction tank 12, the horizontal flow path 13, and the semipermeable membrane M, it is the same as what was shown in FIG.1 and FIG.2. The collecting means for the filtrate F includes a collecting bottom plate 41 and a spacer 45 placed on the collecting bottom plate 41. The recovery bottom plate 41 has a rectangular shape in plan view, and has a dish shape with a flange 42 formed at the peripheral edge. A discharge port 44 for the filtrate F is formed at the center of the bottom plate 41. Further, projections 43 for receiving the spacer 45 are formed on the inner sides of the left and right ends of the bottom plate 41 so as to continue to the flange 42. As shown in FIG. 5, the spacer 45 is a rectangular plate in plan view, and a large number of holes 46 through which the filtrate F passes are formed. The spacer 45 is supported on the protrusion 43 of the recovery bottom plate 41, and there is a space between the spacer 45 and the upper surface of the bottom plate 41. A semipermeable membrane M is supported on the spacer 45.
[0047]
In the synthesis reaction tank 10, the area of the bottom surface of the horizontal flow passage 13 is large, and the semipermeable membrane M is not only the bottom surface of the horizontal flow passage 13 but also the bottom surface of the first reaction tank 11 and the second reaction tank 12. Since it is also arranged on the bottom surface, the area of the semipermeable membrane M becomes very large, and the efficiency of filtration becomes higher. The filtrate F that has passed through the semipermeable membrane M is recovered by the recovery bottom plates 31 and 41 and discharged from the discharge ports 35 and 44. Since the synthesis reaction tank 10 has the horizontal flow passage 13, there is an advantage that the apparatus configuration is simple.
[0048]
FIG. 6 is a perspective view of another example of the synthesis reaction tank. FIG. 7 is a vertical sectional view taken along line VII-VII in FIG. In the following description, the left and right refer to the left as left and the right as the right on the basis of FIG. 7, and the front and rear refer to the front side of the plane of FIG. 7 and the back side of the plane of FIG.
[0049]
6 and 7, the synthesis reaction tank 10 includes a first reaction tank 51 into which the cell-free protein synthesis reaction liquid L is placed, and a synthesis reaction communicated with the first reaction tank 51 via the liquid flow path 53. A second reaction tank 52 for containing the liquid L, a semipermeable membrane M, and means for recovering the filtered liquid F that has passed through the semipermeable membrane M are provided.
[0050]
Both the first reaction tank 51 and the second reaction tank 52 are of a cylindrical structure having a rectangular section with an upper opening formed of an optically transparent plastic. The deepest portion of the right side wall 51a of the first reaction tank 51 has an opening 51b extending from the front side to a half region in the front-rear direction. The deepest part of the left side wall 52a of the second reaction tank 52 is opened 52b from the front side to a half region in the front-rear direction. A meandering flow passage 53 is formed which communicates the opening 51b of the first reaction tank 11 and the opening 52b of the second reaction tank 12 and is meandered by the flow path partition plate 54 along the vertical plane. The meandering flow path 53 is formed to have the same height as the first reaction tank 51 and the second reaction tank 52 so that the area of the side surface thereof is increased. The bottom wall, the front wall, the top wall, and the partition plate 54 of the meandering flow passage 53 are formed integrally with the first reaction tank 51 and the second reaction tank 52.
[0051]
The semipermeable membrane M is vertically arranged over the entire rear side surface of the meandering flow passage 53. That is, the rear side surface of the meandering flow passage 53 is defined and formed by the semipermeable membrane M. The semipermeable membrane M is exchangeable.
[0052]
On the back side of the semipermeable membrane M, a collection chamber 55 for the filtrate F that has passed through the semipermeable membrane M is formed. The rear side wall 55a of the recovery chamber 55 is formed integrally with the first reaction tank 51 and the second reaction tank 52. An outlet 56 for the filtrate F is formed in the bottom wall of the recovery chamber 55.
[0053]
In the synthesis reaction tank 10, the area of the side surface of the meandering flow passage 53 is large, that is, the area of the semipermeable membrane M is large, and the filtration efficiency is high. The filtrate F that has passed through the semipermeable membrane M from the flow passage 53 is discharged from the discharge port 56 of the recovery chamber 55.
[0054]
In the synthesis reaction tank 10, the semipermeable membrane M is disposed only on the rear side surface of the meandering flow passage 53. When the semipermeable membrane M is also disposed on the front side surface of the meandering flow passage 53, the area of the semipermeable membrane M is doubled and the filtration efficiency is further increased.
[0055]
In any of the examples of the synthesis reaction tank described above, the synthesis reaction tank 10 has two reaction tanks, that is, a first reaction tank 11, 51 and a second reaction tank 12, 52. However, in order to move and / or swing the synthesis reaction solution, it may be possible to provide three or more reaction vessels communicated with each other through a liquid flow path.
[0056]
Next, the cell-free protein synthesizer of the present invention provided with means for moving and / or shaking the synthesis reaction solution in the synthesis reaction tank described above will be described. FIG. 8 is a diagram showing a schematic configuration of the cell-free protein synthesizer of the present invention. In FIG. 8, the synthesis reaction tank illustrated in FIGS. 1 to 5 having the horizontal flow passage 13 is drawn for convenience. However, even if the synthesis reaction tank illustrated in FIGS. 6 to 7 having the meandering flow passage 53 is used, a cell-free protein synthesizer can be constructed in exactly the same manner.
[0057]
Referring to FIG. 8, the cell-free protein synthesizer includes a first reaction tank 11, a second reaction tank 12, a liquid flow path 13, a semipermeable membrane M, and a filtrate outlet 35, The moving and / or swinging means 60 of the synthesis reaction liquid L is provided.
[0058]
The moving and / or swinging means 60 includes a syringe pump 61 having two pressurizing ports 61A and 61B and having a plunger 62, and a first pipe 64 communicating from the one pressurizing port 61A to the first reaction tank 11. And an upper sealing cap 65 of the first reaction tank 11 through which the first pipe 64 penetrates, a second pipe 66 leading from the other pressurization port 61B to the second reaction tank 12, and a second pipe 66 to penetrate. The upper seal cap 67 of the second reaction tank 12 and a three-way valve 68 interposed in the middle of the second pipe 66 are mainly configured. The first pipe 64 is provided with a pressure sensor 69. The sealing cap 65 and the sealing cap 67 are movable up and down, and the vertical movement is automatically controlled.
[0059]
A synthesis and filtration operation using this synthesizer will be described. Initially, the three-way valve 68 is closed on the second reaction tank 12 side. The cell-free protein synthesis reaction solution L is placed in the first reaction tank 11 and the second reaction tank 12. Subsequently, the sealing cap 65 and the sealing cap 67 are lowered, and the upper portions of the first reaction tank 11 and the second reaction tank 12 are sealed. The plunger 62 of the syringe pump 61 is actuated to the left, and the pressure in the first reaction tank 11 is increased through the first pipe 64 from the pressure port 61A. At this stage, pressure is applied to the synthesis reaction liquid L, and a part of the synthesis reaction liquid L moves from the first reaction tank 11 side to the second reaction tank 12 side. Thereafter, the atmosphere side of the three-way valve 68 is closed, and the plunger 62 of the syringe pump 61 is reciprocated left and right in a state where the inside of the first reaction tank 11 is pressurized. As a result, the synthesis reaction liquid L is filtered by the semipermeable membrane M while continuously moving or shaking between the first reaction tank 11 and the second reaction tank 12 in a pressurized state. A filtrate containing components that have passed through the semipermeable membrane is obtained from the outlet 35 and the synthesis reaction liquid L is concentrated. By moving and swinging between the first reaction tank 11 and the second reaction tank 12, concentration polarization of the synthesis reaction liquid is suppressed, and filtration is performed in a pressurized state, thereby achieving high-efficiency filtration. Is done. Movement and swinging are controlled by the degree of pressurization and the reciprocating speed of the plunger 62.
[0060]
FIG. 9 shows a modification of the moving and / or swinging means. Referring to FIG. 9, the cell-free protein synthesizer comprises a first reaction tank 11, a second reaction tank 12, a liquid flow path 13, a semipermeable membrane M, and a filtrate outlet 35, The moving and / or swinging means 70 for the synthesis reaction liquid L is provided.
[0061]
The moving and / or swinging means 70 has one pressurizing port 71A and a syringe pump 71 provided with a plunger 72, a pipe 73 communicating from the pressurizing port 71A to the first reaction tank 11, and the pipe 73 passing therethrough. The upper seal cap 74 of the first reaction tank 11 and the upper seal cap 75 of the second reaction tank 12 are mainly configured. The pipe 73 is provided with a pressure sensor 76. The seal cap 74 can move up and down, and the up and down movement is automatically controlled.
[0062]
A synthesis and filtration operation using this synthesizer will be described. The cell-free protein synthesis reaction liquid L is put into the first reaction tank 11 and the second reaction tank 12, and then the sealing cap 74 and the sealing cap 75 are lowered, so that the first reaction tank 11 and the second reaction tank 12 Seal the top. Alternatively, first, the sealing cap 75 is lowered to seal the upper portion of the second reaction tank 12, and then the cell-free protein synthesis reaction solution L is put into the first reaction tank 11, and the sealing cap 74 is lowered to perform the first reaction. The upper part of the tank 11 may be sealed. The inside of the second reaction tank 12 is completely sealed.
[0063]
The plunger 72 of the syringe pump 71 is actuated downward to increase the pressure in the first reaction tank 11 from the pressurizing port 71A through the pipe 73. At this stage, pressure is applied to the synthesis reaction liquid L, and a part of the synthesis reaction liquid L moves from the first reaction tank 11 side to the second reaction tank 12 side. In a state where the inside of the first reaction tank 11 is pressurized, the plunger 72 of the syringe pump 71 is reciprocated up and down. When the plunger 72 operates downward, the air in the first reaction tank 11 is compressed, a part of the synthesis reaction liquid L moves from the first reaction tank 11 side to the second reaction tank 12 side, and is sealed. The air in the second reaction tank 12 is compressed. When the plunger 72 returns upward, a part of the synthetic reaction liquid L moves from the second reaction tank 12 side to the first reaction tank 11 side due to the reaction of the compressed air in the sealed second reaction tank 12. To do. In this way, the semi-permeable membrane moves continuously or swings between the first reaction tank 11 and the second reaction tank 12 in a state where the processing liquid L is pressurized by the reciprocation of the plunger 72 up and down. Filtration is performed by M, and a filtrate containing components that have passed through the semipermeable membrane is obtained from the discharge port 35, and the synthesis reaction liquid L is concentrated. By moving and swinging between the first reaction tank 11 and the second reaction tank 12, concentration polarization of the synthesis reaction liquid is suppressed, and filtration is performed in a pressurized state, thereby achieving high-efficiency filtration. Is done. Movement and swinging are controlled by the degree of pressurization and the reciprocating speed of the plunger 72.
[0064]
For the cell-free protein synthesis system of the present invention, known cell-free protein synthesis systems such as Escherichia coli, embryo, rabbit reticulocyte can be used. Germs such as wheat, barley, rice, corn, spinach, and E. coli can be preferably used. These are prepared by known methods as cell extracts for cell-free protein synthesis.
[0065]
In the present invention, the cell-free protein synthesis reaction solution contains ribosomes, mRNA, substrate amino acids, ATP and GTP. The cell-free protein synthesis reaction solution is placed in the first reaction tank and the second reaction tank to start the synthesis reaction. The addition of each component of the cell-free protein synthesis reaction solution to the synthesis reaction tank is not limited, and various forms are possible.
[0066]
Components that can pass through the semipermeable membrane include degraded products formed during synthesis, ie, ADP, AMP, GDP, GMP, phosphate, pyrophosphate, and the like. These components are inhibitors of protein synthesis and are removed from the synthesis reaction solution through a semipermeable membrane while maintaining a cell-free synthesis system. In addition, the component that can pass through the semipermeable membrane may include a synthesized relatively small target protein.
[0067]
Using the cell-free protein synthesizer of the present invention, the removal operation of the component that can pass through the semipermeable membrane can be performed at a high filtration rate by moving and / or shaking the synthesis reaction solution under pressure. It can be carried out.
[0068]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the filtration efficiency of the component which can pass a semipermeable membrane is high, and a compact cell-free protein synthesizer is provided. In addition, according to the present invention, there is provided a cell-free protein synthesis method that is capable of obtaining a high protein synthesis amount in a short time using the protein synthesizer and that is compact and simple.
[Brief description of the drawings]
FIG. 1 is a perspective view of an example of a synthesis reaction tank in a cell-free protein synthesizer of the present invention.
FIG. 2 is a vertical sectional view of the synthesis reaction tank of FIG.
FIG. 3 is a perspective view of a filtrate collecting bottom plate.
FIG. 4 is a vertical cross-sectional view of a synthesis reaction tank showing a modification of the filtrate recovery means.
FIG. 5 is a perspective view of a spacer.
FIG. 6 is a perspective view of another example of the synthesis reaction tank in the cell-free protein synthesizer of the present invention.
7 is a vertical sectional view taken along line VII-VII in FIG.
FIG. 8 is a diagram showing an example of a schematic configuration of a cell-free protein synthesizer of the present invention.
FIG. 9 is a diagram showing another example of the schematic configuration of the cell-free protein synthesizer of the present invention.
[Explanation of symbols]
10: Synthesis reactor
11: First reactor
13: Liquid flow path
12: Second reactor
L: Synthesis reaction solution
M: Semipermeable membrane
35: Filtrate outlet
60: Means for moving and / or swinging the synthesis reaction liquid L
61: Syringe pump
62: Plunger
64: First pipe
65: Sealing cap
66: Second pipe
67: Sealing cap
68: Three-way valve
69: Pressure sensor

Claims (14)

A first reaction tank containing a cell-free protein synthesis reaction liquid; a second reaction tank containing the synthesis reaction liquid communicated with the first reaction tank via a liquid flow path; a first reaction tank; a liquid A synthetic reaction vessel comprising a semipermeable membrane provided in at least one of the flow path and the second reaction vessel;
Means for moving and / or swinging the synthesis reaction liquid placed in the first reaction tank and the second reaction tank through the liquid flow path ;
A cell-free protein synthesizer provided with a means for collecting a filtrate that has passed through a semipermeable membrane by filtration, and a discharge port for the filtrate collected by the means for collecting, provided in contact with the semipermeable membrane . The cell-free protein synthesizer according to claim 1, wherein the liquid flow path is a substantially horizontal flow path extending horizontally from the deepest part of the first reaction tank to the deepest part of the second reaction tank. The cell-free protein synthesizer according to claim 2 , wherein the semipermeable membrane is disposed so as to define a bottom surface of at least one of the first reaction tank, the substantially horizontal flow path, and the second reaction tank. The fluid channel is a serpentine flow passage extends to the deepest portion of the second reaction vessel while meandering along a substantially vertical plane from the deepest portion of the first reaction vessel, acellular according to claim 1 Protein synthesizer. The cell-free protein synthesizer according to claim 4 , wherein the semipermeable membrane is disposed so as to define at least one of the vertical both sides of the meandering flow passage. The cell-free protein synthesizer according to any one of claims 1 to 5 , wherein the semipermeable membrane is exchangeably provided. Semipermeable membrane, a dialysis membrane, a semipermeable membrane, an ultrafiltration membrane is selected from the group consisting of ultrafiltration barrier, and chemically or biologically film can be captured target substance, of claims 1-6 The cell-free protein synthesizer according to any one of the above. The first reaction tank and / or the second reaction tank is capable of optically measuring absorption, fluorescence, or scattered light of the synthetic reaction liquid, and / or the amount of the synthetic reaction liquid is optical. The cell-free protein synthesizer according to any one of claims 1 to 7 , wherein the cell-free protein synthesizer is configured to be able to be confirmed. It said moving and / or moving means of the synthesis reaction solution is at least one of the interior pressurizing apparatus of the first reaction vessel and second reaction vessel, any of claims 1-8 1 The cell-free protein synthesizer according to Item. The means for moving and / or swinging the synthetic reaction solution includes a syringe pump having two pressure ports and a plunger, a first pipe communicating from the one pressure port to the first reaction tank, A cap for sealing the upper part of the first reaction tank through which one pipe is passed, a second pipe leading from the other pressure port to the second reaction tank, and an upper seal of the second reaction tank through which the second pipe is passed. The cell-free protein synthesizer according to any one of claims 1 to 9 , mainly composed of a cap and a three-way valve interposed in the middle of the second pipe. The means for moving and / or swinging the synthetic reaction solution includes a syringe pump having one pressurizing port and a plunger, a pipe leading from the pressurizing port to the first reaction tank, and penetrating the pipe. The cell-free protein synthesizer according to any one of claims 1 to 9 , mainly composed of an upper sealing cap for the first reaction tank and an upper sealing cap for the second reaction tank. Using the cell-free protein synthesizer according to any one of claims 1 to 11 ,
Put the cell-free protein synthesis reaction solution in the first reaction tank and the second reaction tank, perform the protein synthesis reaction,
A component that can pass through the semipermeable membrane is filtered by moving the synthesis reaction liquid placed in the first reaction tank and the second reaction tank to each other and / or swinging through the liquid flow path. A cell-free protein synthesis method for removing from a synthesis reaction system. The cell-free protein synthesis method according to claim 12 , wherein filtration of components that can pass through the semipermeable membrane is performed under pressure. The cell-free protein synthesis method according to claim 12 or 13 , wherein the component capable of passing through the semipermeable membrane includes a degraded product formed during synthesis.

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