A method of producing polypeptides in a cell-free system

 

(57) Abstract:

The method of synthesis of polypeptides in eukaryotic and prokaryotic cell-free systems based on a modification of the methods of synthesis using cellular lysate or extract in continuous flow or continuous exchange, in which in addition to maintaining the synthesis process due to the reaction mixture of components that support the synthesis and conclusion of the reaction mixture of low molecular weight components, inhibiting the synthesis, continuously changing the concentration of one or more components selected from the group of Mg+2TO+, NTP, polyamines or their combinations and which determine the efficiency of synthesis in a given range of concentrations. The method does not require the use of expensive enzyme exogenous T7 polymerase, which determines the appropriate efficiency of the products of synthesis. 6 C.p. f-crystals, 13 ill., 6 table.

The invention relates to the field of molecular biology, in particular to the synthesis of proteins and polypeptides in cell-free systems derived from prokaryotic and eukaryotic cells.

Synthesis of polypeptides and proteins in cell-free systems is in terms of the constancy of the parameters of the concentrations of Mg2+TO+and temperature. These systems use extracts of prokaryotic (Zubay, 1973) and eukaryotic cells (Roberts and Paterson, 1973; Pelham and Jackson, 1976), as well as natural or synthetic mRNA (Palmenberg, 1990).

In systems of the second generation (Spirin et al., 1988) continuous input substrates (CFCF) feeding the mixture and the output of low molecular weight products, inhibiting the work of the cell-free system, increases uptime and yield of the target protein in comparison with the classical synthesis system in a static (batch) conditions. Well-known works, which were aimed at optimizing the conditions of protein synthesis in CFCF mode (Baranov and Spirin, 1993; Ryabova et al., 1998).

Simultaneously with the improvement of the broadcast system work continued on the improvement of methods of obtaining mRNA in transcription systems, including RNA polymerase and DNA. In these systems, obtaining mRNA depends on the concentration of RNA polymerase and DNA, as well as on the concentration of Mg2+pH, NTP and other ionic conditions (Kern and Davis, 1997). The cost components for in vitro transcription, including RNA polymerase, DNA and NTP is large enough. Therefore, it is necessary to analyze the conditions of the transcription and to optimize the process of obtaining mRNA (Gurevich et al., You coupled transcription-translation (Baranov et al, 1989; Baranov et al., 1995; Ryabova et al., 1998) and methods of synthesis in eukaryotic cell-free systems, in which the stages of transcription and translation occur in the same reaction volume (Spirin, 1992; Baranov and Spirin, 1993; Baranov et al., 1997).

For eukaryotic cell-free systems is known (Craig et al., 1993) that the terms transcription and translation are different and to some extent characterized by the value of the concentration of Mg2+and+.

In the patent EP 0593757 (Baranov et al., 1997) have shown the possibility of continuous (CFCF) synthesis of polypeptides in eukaryotic cell-free transcription systems-broadcast for 20 hours. During synthesis, the concentration value of Mg2+is maintained at the required level due to the constancy of the concentration of Mg2+in the supply mix. Because ribonuclease activity in the reaction system is small and the matrix mRNA retain its activity for a long period of time, the reaction mixture was working previously and newly synthesized matrix mRNA and synthesize the target product at a constant concentration of Mg2+.

To ensure that the synthesis was successful, you need to transcriptional component system transcription-translation was made of sinz SP6 or T7 (up to 30,000 units). In the text of the patent indicated that the optimal conditions of synthesis should be chosen in each case. For such a selection is necessary to spend several syntheses in static (batch) mode values with different concentrations of Mg2+and to determine the optimal value of Mg2+for this polypeptide. The optimization process takes time and is quite costly procedure.

Another way to increase the efficiency of the synthesis is based on the principle of continuous exchange (CECF mode) components of the feed mixture with the components of the reaction mixture through a semi-permeable barrier by diffusion process (Alakhov et al, 1995; Davis et al., 1996; Kim and Choi, 1996; Yamane et al., 1998).

In the General case, the above examples describe the methods developed to maintain constant conditions of the synthesis process. Constant conditions of synthesis provide through output low molecular weight products of the synthesis, inhibiting the work of the cell-free system, and input components that support the synthesis. The maintenance of conditions of synthesis was carried out by selection of the same salt concentrations of Mg2+TO+, NTP and other components in the reaction mixture, and feeding the mixture. For surface and optimized the synthesis process. The optimization process takes time and is rather expensive procedures.

The aim of the invention is the improvement of the method of synthesis of the target polypeptides in eukaryotic and prokaryotic cell-free systems. The invention is based on a modification of the methods of synthesis in continuous flow (CFCF) or continuous exchange (CECF), which along with the maintenance of process synthesis at the expense of putting in the reaction mixture of components that support the synthesis and conclusion of the reaction mixture of low molecular weight components, inhibiting the synthesis, continuously changing the concentration of some selected components that determine the effectiveness synthesis (for example: Mg2+TO+, NTP, polyamines or their combinations) in the prescribed range of concentrations.

The invention is illustrated by drawings:

In Fig.1 shows graphs of changes in the concentration of Mg2+for two examples of cell-free systems, in the synthesis of mRNA and the synthesis of polypeptides in continuous exchange (CECF).

In Fig.2 shows a diagram showing the process of changing the concentration of Mg2+continuous flow (CFCF) when change the calculations of the values.

In Fig.3 shows a graph of changes in the concentration of Mg2+when the periodic pulse is input additional mixture in the volume of the reaction mixture.

In Fig.4 shows a chart of the periodic changes of Mg2+in accordance with the form of the linear gradient.

In Fig. 5 presents a schematic representation of a reactor with one porous barrier.

In Fig. 6 presents a schematic representation of the reaction module and the direction of threads that are formed on the split output high and low molecular weight fractions (CFCF-BF).

In Fig.7 presents a schematic flow distribution in the silent mode of the target product from the synthesis (CFCF-RP).

In Fig.8 presents a schematic representation of the reactor module and the distribution of flows in the mode, when the first porous barrier plays the role of the distributor streams feeding the mixture and additional mixture and target product remains in the synthesis zone (CFCF-RP).

In Fig. 9 presents a schematic representation of the reactor module and the distribution mode is a periodic change of direction of feed of the feeding mixture alternately through lane is transferase (CAT). Chart P refers to the synthesis of static (batch) mode. Chart R shows the kinetics of the synthesis of the CAT in broadcast mode, and dialysis (CECF).

In Fig.11 presents the kinetics of the synthesis of the CAT in the conditions of combined transcription-translation. Chart S refers to the synthesis of static (batch) mode. Chart T represents the kinetics of the synthesis of the CAT in the combined system of transcription-translation and dialysis (CECF) mode.

In Fig. 12 presents the kinetics of the synthesis of the CAT in the conditions of combined transcription-translation with introduction into the reaction mixture an additional mixture and variable concentration of Mg2+and NTP in the reaction mixture during the synthesis. Chart U refers to the synthesis of static (batch) mode. Chart V represents the kinetics of the synthesis of the CAT in the combined system of transcription-translation and dialysis (CECF) mode.

In Fig. 13 shows a chart comparing the results of experiments (6A - 6g), related to the synthesis of the target polypeptide type CAT.

A list of the symbols used in the drawings:

1. F10, F11, F12 streams feeding the mixture;

2. F20, F21, F22 threads for more mixes;

3. F30, F31, F32 - flows of low molecular weight products of the reaction mixture;

4. F40 - high flow is non synthesis;

6. Positions 1-9 correspond to the inputs and outputs of the reactor module;

7. Position 10-19 - refer to structural elements of the reactor.

List of abbreviations used in the text:

Mg2+- magnesium ion, added as a salt.

TO+- the potassium ion, added as a salt.

NTP - triphosphates selected from the group: ATP, GTP, CTP, UTP.

1. Stages of the synthesis of

Synthesis of polypeptides in General consists of several stages. At the stage of preparation of synthesis first create the reaction mixture on the basis of cell lysate or extract, then prepare the feed mixture and an additional mixture, which includes at least one of the selected components, which determines the efficiency of the synthesis. Then select the operating mode of the reactor, the type of reaction module with the specified number and type of porous barriers. Next, select the volume ratio between the reaction mixture and feeding the mixture and/or choose the flow rate of feed of the mixture through the reaction volume. In the next step, gather the installation for synthesis, which includes at least one reaction module, introducing the reaction mixture and feeding the mixture into the respective reaction zone is drawn into the reaction mixture or to one of the parts of the feed mixture to the synthesis or during synthesis. In the synthesis phase, depending on the mode of operation of the additional mixture is administered once, or periodically, or continuously. In the mode of the preparative synthesis of spent high-molecular components is introduced into the reaction mixture once, or periodically, or continuously. The synthesized product is taken from the reaction mixture at the end of the synthesis or during synthesis. When the selection of the product in the synthesis process carried out analysis of the synthesized product, adjust the parameters that determine the productivity of the system.

2. Preparation of reaction mixtures

Cell-free systems are created on the basis of cell lysates and cell extracts and include all components necessary for protein synthesis and regeneration system NTP, buffer and salts, amino acids. It is known for many types of cell-free systems for the synthesis of polypeptides in prokaryotic and eukaryotic systems (Joyce, 1998).

One of the components of a cell-free transcription system-broadcast should be DNA-dependent RNA polymerase that synthesizes mRNA. Used DNA-dependent RNA polymerase is selected from the class E. coli RNA polymerase or bacteriophobic RNA polymerases. This invention considers numarasi.

3. Conditions for the synthesis of

The proposed method does not require additional experiments involving the measurement of the concentration of Mg2+and resolves the contradiction in the known methods, when determining the optimal values of Mg2+and+in conjugated systems transcription-translation, in which the values of Mg+for optimum transcription and optimum broadcast different. During synthesis, along with enter in the reaction mixture of components that support the synthesis and conclusion of the reaction mixture of low molecular weight components, inhibiting the synthesis, there is a continuous change in the concentration of at least one of the selected components (included in the following group: Mg2+, K+, NTP, polyamine or combinations of these components) from the upper to the lower limit of the setting range.

Choose the upper and lower boundaries of the range depends on the mode of synthesis, parameters of the cell-free extract parameters from the reaction mixture and the parameters of the feed mixture. If Mg2+part of the selected components, the boundaries of the area from which selects all ranges of concentrations of Mg2+for different modes (including the ptx2">

If one of the components of the cell-free system using a DNA dependent RNA polymerase, in the mode of transcription of mRNA selection of the upper and lower bounds for a specific range of changes in the concentration of Mg2+carried out the field values from 2 to 50 mM added Mg2+. If protein synthesis is performed in terms of the transcription-translation, the choice of the upper and lower boundaries of a specific range of changes in the concentration of Mg2+carried out the field values from 2 to 25 mM added Mg2+. When protein synthesis in conditions of translation, the choice of upper and lower bounds of a specific range of changes in the concentration of Mg2+carried out the field values from 0.25 to 25 mM added Mg2+. Possible ways in which the upper and lower boundaries of the range of changes in the concentration of Mg2+is chosen so that in the process of synthesis by changing the magnitude of the concentration change of the synthesis conditions and the transition from one mode (for example, preferential transcription) to another mode (for example the mode of transcription of broadcast or pre-emptive broadcast). The examples above reflect only the field values, which can lie in the concentration of one of the outermost boundaries of the range are determined based on knowledge, relating to the conditions of synthesis in prokaryotic and eukaryotic cell-free systems.

An additional purpose of this invention is to reduce the cost of synthesis of a given amount of polypeptide in eukaryotic cell-free systems. In the known methods the synthesis is carried out in conditions of a very high concentration of expensive T7 polymerase, during continuous flow through the reaction volume of the expensive components of the feed mixture, such as NTP and amino acids (Baranov et al. , 1997). In this invention, the efficiency of synthesis in the transcription - translation increases due to the fact that the introduction into the reaction mixture of the high concentration of Mg2+and NTP at the beginning of the synthesis leads to a decrease in the number of abortive mRNA, which, in turn, leads to lower consumption of ATP, GTP and amino acids during translation stage by reducing abortive synthesis of polypeptides.

Described in this invention, examples of the use of the principle of continuous flow of feed mixture through the reaction volume (CFCF) is focused on reducing costs for the preparative synthesis of the desired polypeptide. The flow of feed mixture and the concentration of selected components are easily adjustable with plavanie continuous exchange (CECF) and simple dialyzers also allow to increase the efficiency of synthesis by maintaining concentrations consumed low-molecular components and simultaneous changes in the concentration of selected components. It is known that the rate of exchange of low molecular weight components between the reaction mixture and feeding the mixture through a dialysis membrane depends on many parameters (area, pore size, and others). This imposes some restrictions on the choice of modes of synthesis and selection of the upper and lower limits of the zone in which change the concentration of selected components. For example, when using CECF is more preferable to carry out the synthesis in a separate modes: transcription, translation, transcription-translation, or in combinations of two modes (e.g., transcription and transcription-translation or transcription-broadcast and broadcast). This is due to the fact that the exchange process due to a low enough speed takes time and may not correspond with the speed that is required for entry required for the synthesis of low-molecular components, and output low-molecular products of synthesis, inhibiting the work of the cell-free system.

In Fig. 1 presents two examples of changes in the concentration of Mg2+in the reaction mixture for different modes of synthesis. The first example refers to the case where it is necessary to carry out the synthesis of mRNA in the mode of transcription. As you can see from the chart (To), u is in the reaction mixture due to the high concentration of Mg2+and NTP conditions are created preferential transcription and mRNA synthesis. In this zone, the initial higher concentration of Mg2+over NTP set when preparing the reaction mixture in the range of up to 10 mM. Further decrease in the concentration provides a transition system to the values of the concentrations of Mg2+and NTP corresponding parameters of the upper boundary of the zone transcription-translation (zone C - D). In the second example (figure L) selected conditions, when the upper limit of the range of concentrations of Mg2+and NTP corresponds to the upper boundary of the zone transcription-translation (zone C - D), and the lower limit of the range coincides with the lower boundary of the broadcast area (D-In). In this case, during the synthesis conditions in the reaction mixture changed from predominantly transcriptional up primarily translational. The parameters of the porous barrier (pore size, size of the membrane type membrane) and the flow rate of feed of the mixture along the surface of the porous barrier must be chosen with consideration of the speed of diffusion and exchange of low molecular weight components of the feed mixture and the reaction mixture and must provide the required exchange rate and changes in the concentrations of Mg2+and NTP for time of synthesis. To choose the top and is the means. On the properties of the reaction mixture is influenced by the percentage in the mixture of extract and supply mix. Setting upper and lower limits of the range in which the process of synthesis of changing the concentration of selected components that regulate the efficiency of cell-free system in different modes.

The continuous flow (CFCF) allows a rapid change of speed and direction of flow of the feed mixture through the reaction mixture and, thus, to regulate the rate of change of concentrations of selected components at different stages of the synthesis. During one experiment you can choose different flow rate feeding the mixture through the reaction mixture. This allows the synthesis of polypeptides in a cell-free system of transcription-translation to independently control the duration of different stages, during which the parameters of the reaction mixture and the concentration of selected components correspond to the periods: preferential transcription, transcription-broadcast and broadcast. Thus, the choice of a particular parameter ranges in which to alter the concentration of selected components, depends on: the purpose of carrying out synthesis (mRNA synthesis or the synthesis of the target polypeptide turn from the parameters of the cell-free extract); the choice of parameters of porous barriers (pore size, area and types of membranes); the possible introduction of additional spent macromolecular components. The value of the upper limit of all the permissible ranges of concentrations of Mg2+(from which to choose operating range), mode (CFCF) for synthesis in the transcription-translation using DNA-dependent RNA polymerase, can be selected within 50 mM of Mg2+. The minimum value of the lower values of the concentrations of Mg2+can be a value of 0.25 mM Mg2+.

In Fig. 2 as an example, presents the dependence of (M) changes in the concentration of Mg2+from time to synthesize the system of transcription - translation. By adjusting the flow rate of feed mixture in the first stage of the synthesis (period t1- t2), it is possible to adjust the quantity of synthesized mRNA and prevent overproduction. High concentration of Mg2+and NTP at the beginning of the first period (t1- t2) allows to reduce the number of costly RNA polymerase due to the fact that the synthesis of mRNA is with less abortive mRNA. The ratio of the concentrations of Mg2+and NTP SEL is at the third stage, the excess of Mg2+over NTP was not less than 0.5 mM.

In the long process of synthesis in the mode of CFCF values of concentrations of selected components change from the upper to the lower level once or periodically. In Fig. 3 is a diagram (N) changes in concentrations of Mg2+and NTP when the periodic pulse is input additional mixture in the volume of the reaction mixture. The entire synthesis is divided into N steps with a duration of t4to t6. Additional mixture is introduced at time t4- t5. The concentration of Mg2+and NTP rise, pass level C and the synthesis conditions in the reaction system, enter the zone A-C, which is preferential transcription of mRNA. Lower concentrations of Mg2+and NTP changes the parameters of the synthesis conditions transcription-translation (zone C-D) to conditions predominant broadcast area (D-B).

Preparative synthesis of the target polypeptides often takes a long time, during which the reaction mixture is injected not only low molecular weight components of the feed mixture, but also impose additional molecular components that support long-term synthesis. In the group of high molecular weight components include: ribosomal fraction, the cell-free EXT components is introduced into the reaction volume once, or continuously, or periodically. Components such as the polymerase and the plasmid is preferable to introduce into the reaction mixture simultaneously with the input maximum concentration of Mg2+and NTP at the stage of transcription. Enter the ribosomal fraction is preferably synchronized with the period of the broadcast.

In Fig. 4 is a diagram (A) changes in concentrations of Mg2+and NTP in the formation during the synthesis of linear concentration gradient of these components. Examples of the formation of linear gradients are widely known and used, for example, in liquid chromatography. It is preferable to use this mode for carrying out preparative synthesis of the target polypeptide in the system of the mRNA. In this mode, the values of the concentrations of Mg2+and NTP, included in the total range of concentrations (E-F) should be correlated with the range in which the values of the concentrations of Mg2+and NTP most close to the optimum of the mRNA. The valid range of concentrations of Mg2+and NTP can be determined for known types of extracts from published sources or from technical descriptions supplied by the firms. Some reduction in the efficiency of translation in the areas prerehabilitation the changes in the concentrations of Mg2+and NTP in accordance with the form of the linear gradient. As in the previously considered case, the whole synthesis is divided into N steps with a duration of t7to t9. In the first period of time t7- t8additional mixture containing high concentrations of Mg2+and NTP, is mixed with the feed mixture so that the concentration of Mg2+and NTP in the total mixture is growing. The total mixture is introduced into the reaction mixture and modifies the synthesis conditions. Simultaneously, it supports the synthesis and outputs from the reaction volume of low-molecular components, inhibiting the synthesis. When the change in the proportion of mixed volumes and the reduction of the input of additional mixture in relation to the supply of the mixture level is decreasing concentrations of Mg2+and NTP in the total mixture. The concentration of Mg2+and NTP in the reaction system is reduced and pass through the area (E-F), which is the maximum synthesis. Enter into the reaction, the amount of additional molecular components that support the synthesis, carried out depending on the synthesis conditions continuously or periodically.

Similarly, you can manage and process preparative tranny in static (batch) mode and methods, in which carry out feeding (fed batch) static systems of transcription without output low molecular weight products (Kern and Davis, 1997), are that: (a) by removing low-molecular components, inhibiting the synthesis of mRNA, extended process of synthesis and increases the yield of mRNA; b) due to the choice of the lower boundary of the range, where the concentration of Mg2+and NTP, you can obtain drugs mRNA under conditions conducive to the further stage broadcast synthesized mRNA without additional cleaning procedures; C) the use of high concentrations of Mg2+(for example, up to 50 mM) leads to lower output abortive or incomplete mRNA molecules and reduces the number of costly RNA polymerase.

4. Reactor module

To ensure the considered modes is possible by appropriately choosing the design of the reaction module. Inside the reaction module through porous barriers create the reaction volume and at least one area of the input components of the feed mixture, additional mixtures of high molecular weight components that support the synthesis and output products, inhibiting the synthesis and fusion products.

In the simplest case, the volume reaktsionnoi mixture and the area with a nourishing environment; (b) mode CFCF, the volume is divided porous barrier to the zone with the reaction mixture, which is injected feed mixture, and the area of selection of fusion products (Alakhov et al., 1995).

Known reactors, in which form three zones (Dzewulski et al., 1992; Alakhov et al., 1995; Bauer et al., 1999): the area of input supply mix, the zone with the reaction mixture and the zone output of the synthesis product. This placement areas were caused by the need to maintain constant conditions of synthesis. In this invention the efficiency of the synthesis is provided, on the one hand, by maintaining in the reaction mixture of constant composition of amino acids and other components, and due to active (CFCF) or passive (CECF) regulation values of concentrations of selected components. This condition largely determines the choice of reactor design, intended for different modes. The largest number of applications provides the design of a reactor with two porous barriers, which form three zones in the reactor volume. The number of zones may be large depending on the design features of the reactor module.

The volume of the reaction mixture depends on the conditions and goals of the synthesis. It is known (Thompson et al., 1v et al., 1997) produced in reactors with a volume of 1.0 ml For the synthesis for research purposes minimum reaction volume is chosen in the range from 50 to 500 ál. Synthesis in preparative quantities of lead in one or more reaction modules with capacity from 500 ál to 10 ml, the Number of reaction volumes included in the reactor may be in the range from 1 to 10 depending on the use of a type of reactor modules.

At each point of the reaction volume should be carried out simultaneously three processes: (a) input supply environment; b) the removal of low molecular weight products, inhibiting the synthesis process; C) formation of temporary changes in the concentrations of selected components that determine the efficiency of the synthesis process. It is preferable module design of the reactor, in which the formation of a thin layer of the reaction mixture in any form. The thickness of the layer is chosen from the condition that a continuous exchange of components of the reaction mixture and feeding the mixture or the flow of the feed mixture through the reaction mixture as well as the removal of low molecular weight products, inhibiting the synthesis must occur during the time for which the synthesis is not reduced below an acceptable level. Reactionary see what ispolzovanie hollow fibers, flat membranes or their combinations form the reaction volume can be either cylindrical, or made in the form of a flat layer with a thickness of 0.1 to 5 mm, an Internal volume of the reaction mixture and feeding the mixture can be mixed with or by creating a circulation of the reaction mixture in a closed loop using a pump (Mozayeni, 1995), or by shaking reactor (Choi, 1997), or by using a magnetic stirrer (Kim and Choi, 1996). The volume of the reaction mixture can be pre-populated with various types of separators or organic fillers and inorganic origin. As such can be used a porous, layered capillary materials selected from the group: a) filters of synthetic polymeric or inorganic materials, (b) porous metals or compositions) of gel structures. Introduction porous materials with a pore size of from 10 μm to 0.1 mm in the reaction mixture can increase the area in which the collision of molecules, which ultimately leads to an increase in the speed of the reactions associated with the synthesis of (Alberts et al., 1983). The group of materials in addition to polymeric materials, inorganic oxides, zeolites (Choi, 1997) may include sorbents used for the HRO is th synthesized polypeptide. The use of porous materials of any type is limited to their chemical activity and inhibition of the synthesis.

Porous barriers such as membranes, hollow fibers and other porous structure should facilitate the exchange of components between the feed mixture and the reaction mixture or to play the role of distributors streams feeding the mixture through the reaction volume. There are no restrictions associated with the simultaneous use in the reactor of porous barriers of different types (membrane, hollow fiber) and different types of materials (solid or solid in combination with a gel-like structures). Porous barriers can be used in the form of single layer or multi-layer structures, including the use of different materials.

See the design options associated with the placement of porous barriers relative to each other, can be modified in other ways based on the known knowledge.

Below are examples of the types of thread formation (feed mixtures additional mixtures or combinations thereof) in the reactor modules for efficient synthesis of continuous exchange (CECF) or continuous flow (CFCF).

oC. Temperature range, preferred to extract wheat, lies in the range from 20 to 26oC, for reticulocyte lysate used range from 28 to 38oC, when working with extract E-coli choose the temperature range from 20 to 38oC. To improve the efficiency of exchange between the feed mixture and the reaction mixture create a tangential flow along the inner and outer membrane surface.

For CECF mode settings porous barrier is chosen from the conditions of the conclusion of the reaction volume, low-molecular components (pore sizes are in the range of up to 30 kD), or one who moves 1 and 2 in the reactor module can be sealed in the process of synthesis or opened and is in two parts, the reaction volume is automatically kept at the same pressure. This allows the synthesis process to enter into the reaction or to supply a mixture of substrates, supports the synthesis of, or in addition to changing the concentration of selected components regardless of the diffusion process. Before the synthesis of choosing the ratio of the volume of the reaction and feed mixtures in the range from 1/5 to 1/100 and accordingly choose the appropriate type of reaction module according to the characteristics of volume, pore size and area of the dialysis membrane.

Synthesis of polypeptides for analytical purposes is carried out in microreactors with a reaction volume of 50 µl. Synthesis of polypeptides in preparative quantities of the reaction volume to 50 ml, imposes its own conditions on the method of synthesis and the design of the reactor. Below are options that can be performed using single-channel and multichannel reactors, including split flows within the reaction volume. Options porous barriers, their parameters, the thickness of the layer of the reaction mixture is largely identical to the previous versions.

Reactor design used in flow-through mode (CFCF), should provide input into the reaction volume is not only feeding a mixture comprising a low molecular weight courier. The latter performs the function of the distributor flow and has a pore size of up to 5000 kD, through which freely penetrates most of the components included in the S30 extract, except ribosomes and educated around her complexes. Examples 2 through 5 relate to the use of the proposed method for the synthesis of polypeptides in continuous flow mode (CFCF), and example 6 relates to the synthesis of polypeptides in continuous exchange (CECF).

Example 2. Synthesis of continuous input feed mixture with separation output streams into fractions containing high and low molecular weight components of the synthesis (CFCF-BF), allows the concentration of the synthesized polypeptide inside of the reaction mixture through independent regulation output streams. In Fig. 6 presents a schematic representation of the reaction module and the direction of threads that are formed on the split output high molecular F40 and low-molecular F30 fractions. The reaction module has a housing 10, two porous barrier 11 and 12, which form the reaction volume 14, located between the inner surfaces of the porous barriers and two zones 15 and 16 for input/output liquid communications that come into contact with the external powerchange entrance in the synthesis process served to: (a) feeding a mixture F10; (b) additional mixture F20; (C) the fraction of high-molecular components F50. The supply of the mixture to the input 1 carried out continuously or periodically. Additional mixture F20 and the fraction of high-molecular components F50 served in the reaction mixture depending on the synthesis conditions once, periodically or continuously. The fraction of high-molecular components F50 injected into the reaction volume independently from the supply of the mixture F10, or high-molecular components are pre-mixed with the feed mixture. The synthesis can be carried out without entering fractions F50. Before synthesis selects the ratio between the amounts entered on the experimental conditions factions feed mixtures additional mixtures and high molecular weight fractions relative to the volume of the reaction mixture and the flow rate of these fractions through the reaction volume. The size of the pores of the first porous barrier 11 selects, on the basis of molecular weight and size of the target polypeptide in the range from 30 to 100 kD, the size of the pores of the second porous barrier 12 is chosen in the range of up to 30 kD. Depending on the selected mode of synthesis choose the ratio of the volume passing through the first and second porous barriers in the range from 1/1 to 1/100.

Example 3. In Fig. 7 PR is the same or different pore size, lying within 30 kD. In this case, the synthesis takes place without displaying a high molecular weight fraction F40 and the target product from the synthesis (CFCF-RP), and flows F31 and F32 contain only low molecular weight components of the synthesis. This mode is used in cases when the synthesized polypeptide is greater than the value of 80-100 kD or when the accumulation of synthesized polypeptide in the reaction volume did not inhibit the synthesis process. The input modes of feeding mixtures additional mixtures and fractions with high molecular weight components is similar to the mode CFCF-BF.

Example 4. Porous barriers can be used as distributors streams feeding the mixture and additional mixtures in cases where the volume, which is introduced in the reaction mixture, filled with porous structures and the stirring of the reaction volume is difficult or impossible.

In Fig. 8 shows the structural diagram of the reactor module and the direction of flow mode CFCF-RP, in which the first porous barrier plays the role of a distributor of flow of feed mixture F10 and additional mixture F20. The size of the pores of the first porous barrier 11 are selected in the range up to 5000 kD. This allows you to enter through the first porous barrier part of the macromolecular fraction componentof include tRNA, enzymes, etc. If necessary ribosomal fraction F52 injected into the reaction volume 14 directly through the liquid inlet 1. The parameters of the second porous barrier are selected within 30 kD. The flow of low-molecular components of the synthesis F30, inhibiting the operation of the system, is output from the output 5.

Example 5. In Fig. 9 shows a schematic flow mode CFCF-RF in which a periodic change of direction of feed of feed medium through the first and second porous barriers. In this mode, designed for long-term synthesis of polypeptides, switching directions feed feed mixture helps to clean the pores of the first 11 and second 12 porous barriers. Synthesis is carried out with or without output high molecular weight products from the reactor volume (pore size of the first and second barriers is chosen the same and lies in the range of up to 30 kD), or in the mode selection part of the synthesized product from the reaction volume (the size of the pores of the first porous barrier select up to 100 kD, and the size of the second porous barrier 30 kD). The flow of high molecular weight components F50 is introduced through the inlet 1 into the reaction volume. In the synthesis process of the form N steps input feed mixture and an additional mixture. Kadimazu and additional mixture is connected to input 2. Streams feeding the mixture F11 and the additional flow of the mixture F21 injected into zone 16, which is formed by the surface of the first porous barrier 11. Through the pores of the first porous barrier supply and additional mixture is introduced into a synthesis zone 14 of the reactor and through the pores of the second porous barrier 12 from the zone of the synthesis of low molecular weight components of the synthesis are shown in zone 15 and further form the stream 32, which derive outside of the reactor through the outlet 5. After the first period, the valves switch and connect the tank with the feed mixture and an additional mixture to the inlet 3, which is associated with the area of 15, which formed the second porous barrier 12. Threads F12, F22 through the pores of the second porous barrier penetrate into the reaction mixture and at the same time cleanse the pores of the second barrier, which closed in the first step of the synthesis. Low-molecular components are removed from the reaction volume through the pores of the first porous barrier and form the flow F31, which is derived from the reactor through the outlet 4. Adjusting the duration of the first and second periods of the input streams feeding and additional mixture in the reaction volume through the first or second porous barriers, changing the volumetric flow ratio F31 and F32.

Example 6. the options combined systems of transcription-translation.

It is known that the transcription of the circular and linear forms of DNA fagbemi the polymerase is carried out under different conditions, in particular with a different concentration of Mg2+. To linear form DNA matrix can be defined as a plasmid, linearized with restriction enzymes and PCR products. When using PCR products there is no need to involve living cells to obtain genetic structures (for example, in the case of expression of genes encoding either unstable or toxic products (Marteyanov et al., 1997). In addition, the use of PCR to obtain the matrix allows faster and more convenient to modify its structure at the genetic level: a) to introduce elements that stabilize the structure of the RNA (e.g., highly structured areas, elements of RQ RNA, transcription terminators, and so on); b) to introduce elements that enhance gene expression (for example, enhancers, not broadcast leaders, etc ); C) to provide input marker coding sequences (for example the input epitopes, Tags for affinity selection, and so on).

The method allows to vary the intensity of the transcription and circular plasmids, which can be for several reasons in superstructural form or in the form relacionado Kohl is

The following is an example of the application of this technique to the case of using a circular plasmids for the synthesis of a CAT in a combined system of transcription-translation, on the basis of an extract from wheat germ.

To compare the effectiveness of the proposed method with conventional methods used several reaction mixtures. On the basis of the first reaction mixture has created a translational mixture and perform the synthesis of the CAT, using previously prepared mRNA. In the second variant was added to the reaction mixture components for transcription of mRNA and created the conditions for the combined transcription-translation. In the third embodiment, in the reaction mixture, prepared for synthesis under conditions of combined transcription-translation, was introduced a mixture of the selected components, which are used Mg2+and NTP. Additional components are introduced into the reaction mixture prior to synthesis.

a) synthesis of a CAT in a cell-free system broadcast.

To prepare the reaction mixture for mRNAs in accordance with the data given in table 1. The mixture contains an extract from wheat germ.

Supplying the mixture prepared in accordance with dandingo bag with a diameter of 8 mm of the company Union carbide Corp. with a working volume of 100 µl. The amount of supply of the mixture was 1 ml For comparing the efficiency of synthesis of the overall reaction mixture was divided into two volumes. 30 μl of the reaction mixture was placed in a microcentrifuge tube and 100 μl was placed in the dialysator. Set the dialysator and microprobing in volume and cooled synthesis was performed at a temperature of 25oC. In the synthesis process of the microtube and the dialysis bag was selected aliquots of 5 µl to determine the kinetics of synthesis in static (batch) mode and continuous exchange (CECF). The number of synthesized polypeptide was determined by the method of deposition of synthesized polypeptide on a glass fiber filter trichloroacetic acid, followed by the account of radioactivity in a liquid scintillation counter. In Fig. 10 shows the kinetics of synthesis. Chart P refers to the synthesis of static (batch) mode. Chart R represents the kinetics of the synthesis of the CAT in translational terms in the CECF mode.

b) synthesis of a CAT in the system combined transcription-translation

To prepare the reaction mixture in accordance with the data given in table 3. A combined system of transcription/translation contains plasmid rat enhancer containing the accordance with the data shown in table 4. To maintain the transcription process in the feed mixture was introduced CTP and UTP.

The synthesis parameters (temperature, volume of the reaction mixture and the feeding of the mixture, the type of dialysator) was chosen identical to that shown in example 6a (CAT synthesis in the system broadcast). The results of the synthesis were analyzed as in example 6a.

In Fig. 11 shows the kinetics of the synthesis of the CAT in the combined system of transcription-translation. Chart S refers to the synthesis of static (batch) mode. Chart T represents the kinetics of the synthesis of the CAT in the conditions of the transcription-translation in the CECF mode.

C) synthesis of a CAT in the system combined transcription-translation with continuous changes in the concentration of Mg2+, NTP in the reaction mixture during synthesis.

The reaction mixture (which before the beginning of the synthesis included an additional mixture consisting of selected components of Mg2+and NTP) was prepared in accordance with the data given in table 5.

Feeding the mixture is prepared in accordance with the data given in table 6.

The synthesis parameters (temperature, volume of the reaction mixture and the feeding of the mixture, the type of dialysator) was chosen similar to that shown in example 6a (CAT synthesis "ptx2">

In Fig. 12 shows the kinetics of the synthesis of the CAT in the combined system of transcription-translation, in which at the beginning of the synthesis included an additional mixture of the selected components, Mg2+and NTP. Chart U refers to the synthesis of static (batch) mode. Chart V represents the kinetics of the synthesis of the CAT in the reaction mixture, which before the beginning of the synthesis was introduced a mixture of Mg2+and NTP and spent the synthesis conditions combined transcription-translation in the CECF mode with variable concentrations of Mg2+and NTP in the reaction mixture during synthesis.

In Fig. 13 is a chart that compares the results of experiments synthesis of the target polypeptide CAT. Data are taken from examples 6 and in and represent the output of CAT (µg/ml) in different modes: a) static (batch) mode in the conditions of combined transcription-translation (example 6b, lane W), b) broadcast (example 6a, band X), C) combined transcription-translation (example 6b, lane Y), g) combined transcription-translation with variable parameters concentration Mg2+and NTP in the reaction mixture (example 6b, lane Z). Comparison of the results shows that the highest yield of polypeptide CAT (32 μg/ml) obtained by the combined transcription-Tran is eenie can be used in research and applied practice for the synthesis of polypeptides in a cell-free system based on extracts of eukaryotic and prokaryotic cells. The described method allows the researcher to optimize the entire process of synthesis in General, but to investigate the contribution of individual components in maintaining a synthesis at different stages of transcription, transcription-broadcast and broadcast.

Sources of information

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Baranov, V. I. et al. Method for preparative expression of genes in a cell-free system of conjugated reduced/translation. EP Patent 0401369, Int. Cl. C12P 21/00 (31.05.1995).

Baranov, V. I., A. S. Spirin Gene expression in cell-free system on preparative scale. From: Methods in Enzym." Vol. 217 "Recombinant DNA", Part H, Edit. R. Wu, Academic Press p.p. 123-142 (1993).

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Choi, S. et al. Method producing protein in a cell-free system. US Patent 5593856, US Cl. 435/68.1 (14.01.1997).

Craig D. et al. Plasmid cDNA - directed protein synthesis in a coupled eukaryotic in vitro reduced-translation system, Nucl. Acids Res. V. 20, No. 19: 4987-4995 (1993).

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1. A method of producing polypeptides in a cell-free system, comprising preparing a reaction mixture with the use of cell lysate or extract, which selects the mode of synthesis, establish the parameters of the cell-free system, determine the mode of exchange of components between the reaction and the feed mixtures, choose the type and parameters of the at least one porous barrier, enter the reaction and the supply of the mixture in the reaction module and carry out the synthesis, characterized in that the step of selecting the mode of synthesis determines the type of at least one of the selected components, a cell-free system, which affects the efficiency of synthesis, establish upper and lower bounds of the range of concentrations of selected components, carry out the synthesis under conditions when the concentration of selected components change once, periodically, or continuously in the selected range.

2. The method according to p. 1, characterized in that at least one of the selected components included in the group consisting of Mg2+and NTP in excess of the values of Mg2+over NTP.

4. The method according to p. 1, characterized in that the mode of synthesis is chosen from the group including transmission, coupled transcription-translation, transcription-translation carried out in a single volume, transcription, or a combination of these modes.

5. The method according to p. 4, characterized in that depending on the mode of synthesis of NTP, which is included in the reaction mixture, are a group of ATP, GTP, UTP, CTP or ATP, G.

6. The method according to p. 1, characterized in that the exchange mode is chosen from the group of continuous exchange, continuous flow, or combinations thereof.

7. The method according to p. 1, characterized in that the reaction mixture is prepared on the basis of cell-free systems from cells prokaryotes or eukaryotes or combinations thereof.

 

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49 cl, 5 dwg, 2 tbl, 9 ex

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