RU199182U1 - DEVICE FOR PROTEIN SYNTHESIS IN A CELLLESS SYSTEM - Google Patents

Abstract

The utility model relates to the field of molecular biology and biotechnology. The device for the synthesis of proteins in a cell-free system contains a reaction cell with the first and second porous barriers forming a volume with the reaction mixture. The pumps provide the supply of the feed mixture solution into the reaction volume and the removal of the solution of the synthesis products. The device additionally contains a liquid communication external with respect to the reaction cell, which connects the inlet and outlet of the reaction volume by means of the first and second valves and a pump connected in series. This fluid communication allows for partial or complete change of the reaction mixture in the cell and prolong the efficient synthesis of the target proteins. 6 c.p. f-ly, 1 dwg

Description

The utility model relates to molecular biology and biotechnology, and specifically to devices for the synthesis of polypeptides and proteins in cell-free systems.

State of the art

The considered utility model relates to devices for the synthesis of polypeptides and proteins in cell-free systems. The designs of devices for cell-free synthesis are designed with the possibility of increasing the efficiency of synthesis.

One of the ways to increase the efficiency of synthesis is based on the use of the principle of continuous exchange (CECF mode) of the components of the feeding mixture with the components of the reaction mixture of the cell-free system through a semipermeable barrier made of dialysis membrane due to the diffusion process (Choi et al. 1997; Yamane et al., 1998, Endo et al., 2005). Devices that implement the principle of continuous exchange of the components of the feed mixture with the components of the reaction mixture are simple to manufacture, but do not provide the possibility of synthesizing a sufficient amount of protein.

To increase the efficiency of the synthesis, devices were developed that implemented the (CFCF) principle, which consists in the formation of a continuous withdrawal of the reaction products from the reaction mixture and the continuous restoration of the initial concentration of low molecular weight substances consumed in the synthesis process (Alakhov Yu.B. et al. 1991, Spirin AS 1992, Alakhov et al., 1995, Biryukov et al., 2000, Biryukov et al. 2001).

The closest to the claimed device are variants of the device for the synthesis of proteins and polypeptides in a cell-free system, taken as a prototype described in the patent of the Russian Federation No. 2148649. This patent describes variants of the synthesis of one type of polypeptide or protein in one or more parallel connected reaction volumes. In this case, the conditions for carrying out the synthesis in one reaction volume or with parallel connection of several reactors are strictly the same, they have the same type of feed mixture and a similar composition of active elements performing translation or transcription / translation in the process of biosynthesis. An increase in the efficiency of synthesis occurs due to the summation of the synthesized products synthesized in several reaction volumes.

In the known devices, it is difficult to increase the time of protein biosynthesis due to the fact that, with the time of biosynthesis, the components of the cell-free system that are not included in the feed mixture are washed out of the reaction volume.

There is a need to develop new devices that make it possible to increase the yield of the target product not due to the parallel connection of several reactors, but due to an increase in the synthesis time in one volume of the reaction cell. The task of developing a useful model is to provide a researcher with the possibility of producing a preparative amount of protein in the process of cell-free synthesis.

The purpose of the development of the utility model is to improve the device for the synthesis of target polypeptides in eukaryotic and prokaryotic cell-free systems. The technical solutions given in the utility model are associated with a modification of the synthesis method in continuous flow mode (CFCF), in which, along with maintaining the synthesis process by introducing components into the reaction mixture that support the synthesis and removing low molecular weight components that inhibit the synthesis from the reaction mixture, periodic partial or complete change of the reaction mixture.

The technical result to be achieved by the claimed utility model is to increase the efficiency of the reactor and to ensure the duration of the synthesis process.

The essence of the technical solution to the utility model.

To achieve the above goals, the object of the utility model is a device for the synthesis of proteins in a cell-free system, containing a reaction cell with the first and second porous barriers forming a volume with the reaction mixture, pumps providing the supply of a solution of the feed mixture to the reaction volume and removal of the solution of synthesis products, storage tanks for solutions feeding mixture, high-molecular synthesis products, low-molecular synthesis products. The device additionally contains an external, in relation to the reaction cell, liquid communication, which connects the inlet and outlet of the reaction volume using series-connected first and second valves and a pump. The outlet of the reaction volume is connected to the outlet of the first tap, the first inlet of which is connected to the first inlet of the second tap, the second inlet of the first tap is connected to the tank for collecting the spent reaction mixture, the second inlet of the second tap is connected to the container containing the new reaction mixture. The outlet of the second valve is connected to the inlet of the pump, which allows pumping a new reaction mixture to the inlet of the reaction volume of the cell.

Another aspect of the utility model is associated with the choice of the parameters of the reaction cell, where the reaction volume of the cell is chosen in the range from 100 μl to 10 ml, the parameters of the first porous barrier are selected in the range of 50-300 kDa, preferably, in the range of 50-100 kDa, the pore size of the second porous barrier choose in the range of 10-30 kDa.

The next aspect of the utility model is associated with the choice of synthesis conditions, where the synthesis is carried out in the temperature range from +25 to + 37 ° C, the reaction and feed mixtures are kept in the range from +2 to + 7 ° C.

Brief description of the drawings.

FIG. 1 shows a block diagram of a device for protein synthesis, in which the possibility of partial or complete change of the reaction mixture is realized.

Description of the utility model

In the process of research and optimization of devices that allow for the long-term synthesis of polypeptides and proteins in cell-free systems, it turned out that due to the leaching of some components that make up the reaction mixture, in which cell-free synthesis of polypeptides and proteins occurs, after a certain period of time, the synthesis efficiency decreases, despite for additional feeding of the reaction mixture with low molecular weight components, which are washed out from the reaction mixture in large quantities.

It was found that the replacement in the reaction volume of part or all of the exhausted reaction mixture with a fresh solution of the reaction mixture makes it possible to continue efficient protein synthesis. The change of the reaction mixture in the working volume of the cell can be carried out due to the formation of an external liquid communication, which makes it possible to move a new portion of the reaction mixture into the working volume of the cell.

The device of the utility model is illustrated by an exemplary embodiment with reference to the attached drawing.

To implement this technical solution, a device for the synthesis of proteins in a cell-free system, the structural diagram of which is shown in Fig. 1 consists of a reaction cell (1) in which the first (3) and the second (4) porous barriers are installed, the distance between the inner surfaces of which forms the working volume (2), in which the synthesis of polypeptides and proteins is carried out. Outlets of the pump (10) and pump (14) are connected to the input (6) of the reaction cell (1). The pump (10) delivers the feed mixture placed in the tank (11). The pump (14), together with the taps (15) and the tap (16), is part of the external fluid communication connecting the inlet (6) and outlet (8) of the working volume of the reaction cell (1). This makes it possible to supply to the inlet (6) of the reaction cell (1) through the liquid valve (16) and the pump (14) a partial or full volume of the reaction mixture stored in the vessel (18) for changing the spent reaction mixture in the reaction volume (2) of the cell ( 1).

In addition, a closed external fluid communication connected to the inlet (6) and outlet (8) of the reaction volume (2) through taps (15) and (16) allows longitudinal (along the inner surface) of porous barriers (3) and (4) to move reaction mixture (forward / backward) when changing the reverse of the pump (14). This makes it possible to stir the reaction mixture and partially open the pores of the porous barriers (3) and (4) in cases where the volume of the reaction mixture is small and does not allow introducing a stirrer into the volume (2) of the reaction cell.

The outlet (5) of the reaction cell (1) is connected to the tank (9) for draining the low molecular weight fractions of the reaction mixture containing the decomposition products formed during the synthesis. The outlet (7) of the reaction cell (1) is connected to the inlet of the pump (12), the outlet of which is connected to the reservoir (13), in which the synthesized high-molecular product in the form of polypeptides and proteins is collected for subsequent purification and storage. The outlet (8) of the reaction cell (1) is connected to a liquid tap (15) through which the spent reaction mixture moves from the working volume (2) of the reaction cell (1) to the reservoir of the vessel (17) when filling the volume (2) with the feed mixture coming from the vessel (11) with the pump (10).

The device works as follows. At the first stage, the reaction mixture and the feed mixture are prepared. As an example, which includes, but does not limit the options for the preparation and composition of the reaction mixture and the feed mixture, it is possible to use the concentrations of the components and the composition of the mixtures, as described in RF patent No. 2148649.

The synthesis of polypeptides and proteins in the reaction cell is carried out at a temperature from +25 to + 37 ° C. The feed mixture is fed into the reaction cell at a rate of 1.5-2.0 of the internal volume of the cell per 1 hour. The reaction mixture is fed with a feed mixture. Product selection is carried out. In the process of synthesis, the yield level of the synthesized product is analyzed, for example, by measuring the fluorescence of the solution (Biryukov S.V. et al. 2000).

Determine the time after which the efficiency of the synthesis falls, despite the feeding of the reaction mixture with the feed mixture. The operation of the device is transferred to the mode of partial or complete change of the reaction mixture in the reaction volume. To do this, the supply of the feed mixture is stopped, the pump (14) is turned on and the valve (16) is switched to a position that facilitates the supply of the reaction mixture from the container (18) through the switched on pump (14) to the inlet of the reaction volume (2) of the reaction cell (1). To drain the reaction mixture from the reaction volume (2), switch the valve (15) to the position when from the outlet of the reaction volume (2) the mixture enters the container (17).

The parameters of the first porous barrier are selected in the range of 50-300 kDa, preferably in the range of 50-100 kDa, the pore size of the second porous barrier is selected in the range of 10-30 kDa.

The reaction volume of the cell is selected in the range from 100 μl to 10 ml. Preferred is the design of the reactor module, in which the formation of thin layers of the reaction mixture of any shape is carried out. The thickness of the layer of the reaction mixture is selected in the range from 0.1 to 10 mm from the condition that, given the areas of the first and second porous barriers and the selected pore sizes of the barriers, the average rate of feeding the feed medium into the reaction volume provides the input of the feed mixture components to the most distant points of the reaction mixture during the time during which the concentration of the nutrient medium at remote points decreases to an acceptable level, and the concentration of low molecular weight components that inhibit the synthesis process does not exceed the allowable level.

In this case, the reaction mixture stored in the tank (18) and the feed mixture in the tank (11) are in an additional thermostat with a temperature from +2 to + 7 ° C.

It is preferable to use additional stirring in the zone of the reaction volume. This stirring can be achieved by rotating a magnetic stirrer. Additionally, due to the short-term activation of the pump (12) in the reverse mode during the synthesis, it is possible to clean the pores of the first porous barrier (3), which are clogged during the synthesis.

The advantage of the utility model is that during the operation of the device, its structure allows:

a) free introduction of the nutrient medium into the reaction volumes, b) removal of low molecular weight products that inhibit the synthesis process, c) partial or complete replacement of the reaction mixture to maintain synthesis, d) effective cleaning of pores of porous barriers, e) reduction of dead volumes in liquid communications for injection / withdrawal of high molecular weight and low molecular weight components and synthesis products, f) stirring the reaction mixture.

The proposed utility model improves the efficiency of the device, provides a longer synthesis process and ease of use.

Sources of information

Choi C. et al. Method producing protein in a cell-free system: US Patent 5593856, U.S. Cl. - 435 / 68.1, Jan. 14 (1997).

Yamane T. et al. Synthesis of protein by cell-free protein synthetic system and apparatus therefor. JP Patent 10080295, IPC Cl. C12P 21/00 (31.03.1998).

Endo Y. et al. Preparation containing cell extracts for synthesizing cell-free protein and means for synthesizing cell-free protein US Patent 6905843 IPC Cl. C12M 1/12 (2005-06-14)

Alakhov Y.B. and other Method of obtaining peptides and proteins in a cell-free translation system, Ed. wit. SU 1618761 Al, Int. Cl. С12Р 21/02, 07.01.91, Bull. N1 (1991).

Spirin A.S. Cell-Free Protein Synthesis Bioreactor, from Frontiers in Bioprocessing II, 31-43, Editors: Todd P. et al., Amer. Chem. Soc. (1992).

Alakhov Yu. B. et al. Method of preparing polypeptides in a cell-free translation system: US Patent 5478730, U.S. Cl. - 435 / 68.1, Dec. 26 (1995).

Biryukov S.V. and others. METHOD FOR OBTAINING POLYPEPTIDES IN A CELLLESS SYSTEM (VERSIONS) AND A DEVICE FOR ITS IMPLEMENTATION RF Patent No. 2148649, С12Р 21/00 (10.05.2000).

Biryukov S.V. and others. METHOD FOR OBTAINING POLYPEPTIDES IN A CELLLESS SYSTEM RF Patent 2169154, C07K 1/04 (20.06.2001).

Claims (7)

1. A device for the synthesis of proteins in a cell-free system, containing a reaction cell (1) with the first (3) and second (4) porous barriers, where the reaction volume (2) with the reaction mixture is formed between the inner surfaces of these barriers, storage capacity (11) feed mixture connected through a pump (10), providing the supply of a solution of the feed mixture, with the inlet (6) of the reaction volume (2), a container (13) for storing high-molecular-weight synthesis products connected through a pump (12), providing removal of high-molecular synthesis products in the form polypeptides and proteins, with the outlet (7) of the reaction cell (1), a container (9) for draining low molecular weight fractions of the reaction mixture, connected to the outlet (5) of the reaction cell (1), characterized in that it additionally contains an external in relation to the reaction cell (1) liquid communication, which connects the inlet (6) and outlet (8) of the reaction volume (2) using the first (15) and second (16) taps and pump (14) connected in series, wherein the outlet (8) of the reaction volume is connected to the outlet of the first tap (15), the first inlet of which is connected to the first inlet of the second tap (16), the second inlet of the first tap (15) is connected to the container (17) for collecting the spent reaction mixture, the second the inlet of the second valve (16) is connected to the vessel (18) containing the new reaction mixture, where the outlet of the second valve (16) is connected to the inlet of the pump (14), which provides the possibility of pumping the new reaction mixture into the reaction volume (2) of the reaction cell (1 ). 2. A device according to claim 1, characterized in that the reaction volume (2) of the cell is from 100 μl to 10 ml. 3. A device according to claim 1, characterized in that the reaction volume (2) of the cell contains a magnetic stirrer. 4. The device according to claim. 1, characterized in that the first (3) and second (4) porous barriers are made of flat membranes. 5. A device according to claim 1, characterized in that the pore size of the first (3) porous barrier is 50-300 kDa. 6. A device according to claim 1, characterized in that, preferably, the pore size of the first (3) porous barrier is 50-100 kDa. 7. The device according to claim 1, characterized in that the pore size of the second (4) porous barrier is 10-30 kDa.

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