Method of preparing polypeptides in cell-free system and device for its realization

Abstract

The present invention provides a method for synthesis of polypeptides in a cell-free system. In this method, products of synthesis are separated into a low molecular weight fraction and a high molecular weight fraction, with the high molecular weight fraction containing the target polypeptide. The method calls for the removal of at least a portion of the low molecular weight fraction via a porous barrier.

What is claimed:  
     
         1 . A method for obtaining polypeptides in a cell-free translation system by which the reaction mixture, the feed solution and expendable components of the high molecular weight fraction are prepared, at least two porous barriers are placed inside the reactor module, the reaction mixture is introduced to the reactor volume and incubated at given conditions maintaining the synthesis, the target polypeptide is removed or retained in the reactor volume, wherein products of synthesis are branched in a low molecular weight fraction and a fraction which contains high molecular weight components with the target polypeptide, the main part of the low molecular weight fraction is removed via at least one part of the second porous barrier, the ratio of the volume of the fractions of feed solution and expendable components to the volume of the fraction containing the target polypeptide is chosen, modes of supply of the feed solution and expendable components of the fractions are realized.  
     
     
         2 . The method according to  claim 1  wherein in the mode of removal of the target polypeptide from the reactor volume the fraction containing high molecular weight products with the target polypeptide is removed via the first porous barrier which has pore sizes up to 100 kD, while the low molecular weight fraction with components of the reaction mixture and products of synthesis is removed via at least one part of the second porous barrier which has pore sizes up to 30 kD.  
     
     
         3 . The method according to  claim 1  wherein in the mode when the target product is not removed from the reaction mixture, the fraction of low molecular weight components is removed from the reactor simultaneously via the first and second barriers which have pore sizes up to 30 kD.  
     
     
         4 . The method according to  claim 1  wherein the feed solution is supplied to the reactor volume either (i) via the inlet of the reactor volume, or (ii) via the pores of another part of the second barrier, or (iii) both via the inlet and the pores.  
     
     
         5 . The method according to  claim 2  wherein the fraction of high molecular weight components with the target polypeptide is removed from the reactor continuously or recurrently.  
     
     
         6 . The method according to  claim 1  wherein the fraction of expendable high molecular weight components is added to the reaction mixture either (i) before the synthesis, or (ii) once during the synthesis, or (iii) recurrently during the synthesis, or (iv) is supplied continuously during the synthesis.  
     
     
         7 . The method according to  claim 6  wherein the expendable high molecular weight components are chosen from (i) ribosome fraction, (ii) cell-free system extracts (S30, S100 and their modification), or (iii) polymerases, plasmids, tRNAs that are supplied to the reactor mixed with the feed solution, each of them taken separately or combined with the others.  
     
     
         8 . The methods according to  claim 1  wherein the ratio of the feed solution volume, that is supplied to the reactor, to the volume of the fraction containing high molecular weight components with target polypeptides, that are removed from the reaction mixture, is from 1 to 100.  
     
     
         9 . A method for obtaining polypeptides in a cell-free translation system by which the reaction mixture the feed solution and expendable components of the high molecular weight fraction are prepared, at least two porous barriers are placed inside the reactor module, the reaction mixture is introduced to the reactor volume and incubated at given conditions maintaining the synthesis, the target polypeptide is retained in the reactor, wherein the low molecular weight fraction which consists of removed components including low molecular weight components of the reaction mixture and low molecular weight components of the synthesis is withdrawn from the reaction volume via at least one the second porous barrier only, the mode of supply of fractions of the feed solution and expendable components is realized.  
     
     
         10 . The method according to  claim 9  wherein low molecular weight components of the feed solution are supplied to the reactor volume (i) via the first porous barrier, or (ii) part of the feed solution is supplied to the reactor volume via the inlet while the other part is supplied via the first porous barrier.  
     
     
         11 . The method according to  claim 9  wherein expendable high molecular weight components are supplied either (i) via the reactor volume inlet, or (ii) via the first porous barrier, or (iii) part of the expendable components is supplied via the reactor volume inlet while the other part is supplied via the first porous barrier.  
     
     
         12 . The method according to  claim 9  wherein the first porous barrier has pore sizes up to 1000 kD and the second porous barrier has pore sizes up to 30 kD.  
     
     
         13 . The method according to  claim 9  wherein the fraction of expendable high molecular weight components is added to the reaction mixture either (i) before the synthesis, or (ii) once during the synthesis, or (iii) recurrently during the synthesis, or (iv) is supplied continuously during the synthesis.  
     
     
         14 . The method according to  claim 9  wherein expendable high molecular weight components are chosen from (i) ribosome fraction, (ii) cell-free system extracts (S30, S100 or their modifications) and (iii) polymerases, plasmids and tRNAs that are supplied to the reactor mixed with the low molecular weight feed solution, either taken separately or combined with the others.  
     
     
         15 . A method for obtaining polypeptides in a cell-free translation system by which the reaction mixture the feed solution fraction and expendable components of the high molecular weight fraction are prepared, at least two porous barriers are placed inside the reactor module, the reaction mixture is introduced to the reactor volume and incubated at given conditions maintaining the synthesis, the target polypeptide is removed or retained in the reactor volume wherein during the synthesis N cycles are formed, everyone of which consists of at least two steps, at the first step low molecular weight components of the feed solution are supplied to the reactor via the first porous barrier and the low molecular weight fraction with products of synthesis and components of the reaction mixture is removed via the second porous barrier, at the second step the supply and removal channels are switched and low molecular weight components of the feed solution are supplied via the second porous barrier, the low molecular weight fraction or the high molecular wieght fraction containing products of synthesis and components of the reaction mixture is removed via the first porous barrier, the mode of supply of of fractions of the feed solution and expendable high molecular weight components is realized  
     
     
         16 . The method according to  claim 15  wherein in the mode when the target polypeptide is removed from reaction volume the first porous barrier has pore sizes up to 100 kD and the second porous barrier has pore sizes up to 30 kD.  
     
     
         17 . The method according to  claim 16  wherein in the mode when the target polypeptide is removed the ratio of the feed solution volume supplied to the reactor during N cycles to the fraction volume including the target polypeptide removed from the reaction mixture is from 2 to 100.  
     
     
         18 . The method according to  claim 15  wherein in the mode when the target polypeptide is retained in the reaction volume the first and second barriers have pore sizes up to 30 kD.  
     
     
         19 . The method according to  claim 15  wherein the fraction of expendable high molecular weight components is added to the reaction mixture either (i) before the synthesis, or (ii) once during the synthesis, or (iii) recurrently during the synthesis, or (iv) is supplied continuously during the synthesis.  
     
     
         20 . The method according to  claim 15  wherein expendable high molecular weight components are chosen from (i) ribosome fraction, (ii) cell-free system extracts (S30, S100 or their modifications), or (iii) polymerases, plasmids and tRNAs that are supplied to the reactor, each taken separately or combined with the others.  
     
     
         21 . The method according to  claim 15  wherein the duration of the first and second steps in every of the N cycles is adjusted depending on the conditions of synthesis and the changes in its parameters.