Method for producing ribosome in cell-free protein synthetic line

Abstract

The purpose of the present invention is to provide a method for producing a ribosome large subunit by a one-pot reaction in a cell-free protein synthetic system. A method for producing a ribosomal large subunit in a cell-free protein synthetic line, said method comprising: a step for assembling a ribosomal large subunit protein and a ribosomal large subunit RNA in a cell-free protein synthetic line to form a ribosomal large subunit; at least one of the ribosomal large subunit proteins is generated from a template nucleic acid encoding a ribosomal large subunit protein.

Description

Technical Field

[0001] The present invention relates to a method for producing a large ribosomal subunit and / or a small ribosomal subunit by a one-pot reaction in a cell-free protein synthesis system. Background Art

[0002] Ribosomes are molecular complexes that read genetic information from mRNA and synthesize proteins. Ribosomes are composed of small subunits (SSU) and large subunits (LSU). For example, the 70S ribosome of Escherichia coli is composed of a 30S subunit (SSU) composed of 21 proteins and one RNA (16S rRNA), and a 50S subunit (LSU) composed of 33 proteins and two RNAs (23S rRNA and 5S rRNA).

[0003] If the catalytic activity of the ribosome can be changed by introducing mutations into the constituent elements of the ribosome, it is expected that the translation efficiency of the protein will be improved, the efficient synthesis of non-natural proteins containing non-natural amino acids, and the efficient synthesis of non-natural polymers containing ester bonds, thioester bonds, glycosidic bonds, etc. can be achieved. However, the ribosome is an essential factor for life, and the introduction of mutations into the ribosome is directly related to cell death in most cases, so it is extremely difficult to produce cells containing ribosomes into which mutations have been introduced. Therefore, in the past, a technology for reconstructing ribosomes in vitro has been developed.

[0004] For example, Non-Patent Document 1 reports that ribosomal RNA (rRNA) is transcribed in vitro in the presence of all ribosomal proteins (r-proteins) separated from 70S ribosomes, thereby reconstructing ribosomes using the transcribed rRNA and r-proteins. This method has the disadvantage that it is easy to introduce mutations into rRNA, but it is not possible to introduce mutations into r-proteins.

[0005] In addition, Non-Patent Document 2 reports that: in vitro, in the presence of 20 SSU r-proteins, SSU can be reconstructed by simultaneously performing transcription of 16S rRNA and transcription and translation of 1 SSU r-protein. This method requires the expression and purification of 20 SSU r-proteins for preparation, and therefore has the disadvantage of requiring a lot of time and labor. In addition, although mutations can be introduced into rRNA and 1 SSU r-protein to be transcribed and translated, in order to introduce mutations into the 20 SSU r-proteins directly added to the reaction system, it is necessary to prepare mutants of r-proteins separately, and there is a disadvantage that ribosomes into which mutations have been introduced into more than two r-proteins cannot be easily produced. Producing r-proteins from template nucleic acids to produce ribosomes requires highly coordinated transcription, translation, and assembly, and is therefore extremely difficult. In Non-Patent Document 2, the generation of more than two SSU r-proteins from template nucleic acids is not achieved. Furthermore, it is known that ribosomal LSU has a complex structure and is difficult to reconstruct compared to ribosomal SSU, and therefore it is not considered possible to apply the technology of Non-Patent Document 2 related to the reconstructing of ribosomal SSU to the reconstructing of ribosomal LSU.

[0006] Prior art literature Non-patent literature Non-patent literature 1: Michael C Jewett et al., In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation, Mol Syst Biol. 2013 Jun 25; 9: 678. doi: 10.1038 / msb.2013.31. Non-patent document 2: Shimojo, M. et al. In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomalelements in E. coli. Commun Biol 3, 142 (2020). Summary of the invention

[0007] Technical problem to be solved by the invention An object of the present invention is to provide a method for producing ribosomal LSU, ribosomal SSU or ribosome by a one-pot reaction in a cell-free protein synthesis system.

[0008] Technical solutions for solving technical problems The present inventors conducted intensive studies to establish a technology for producing ribosomal LSU by a one-pot reaction in a cell-free protein synthesis system, and as a result, found that in a method for producing ribosomal LSU including a step of assembling LSU r-protein and LSUrRNA in a cell-free protein synthesis system to form ribosomal LSU, by causing at least one of the LSU r-proteins to be produced from a template nucleic acid in the cell-free protein synthesis system, ribosomal LSU can be easily produced by a one-pot reaction.

[0009] In addition, the present inventors conducted intensive studies to establish a technology for producing ribosomal SSU by a one-pot reaction in a cell-free protein synthesis system, and as a result, found that: in a method for producing ribosomal SSU including a step of assembling SSU r-protein and SSU rRNA in a cell-free protein synthesis system to form a ribosomal SSU, by causing at least two of the SSU r-proteins to be produced from a template nucleic acid in the cell-free protein synthesis system, the ribosomal SSU can be easily produced by a one-pot reaction.

[0010] Furthermore, the present inventors conducted intensive studies to establish a technology for producing ribosomes by a one-pot reaction in a cell-free protein synthesis system, and as a result, found that in a method for producing ribosomes including the steps of forming ribosomes by assembling LSU r-protein and LSU rRNA and assembling SSU r-protein and SSU rRNA in a cell-free protein synthesis system, ribosomes can be easily produced by a one-pot reaction by producing at least one of LSU r-protein and SSU r-protein from a template nucleic acid in the cell-free protein synthesis system.

[0011] In addition, since mutations can be easily introduced into the template nucleic acid of the r-protein by conventional methods, in the above-mentioned method using the template nucleic acid of the r-protein as a starting material, by adding mutations to the template nucleic acid, ribosomal SSU and / or LSU into which mutations are introduced can be easily produced. The present invention was completed through further studies based on these findings.

[0012] In one embodiment of the present invention, there are provided a method for producing a ribosomal LSU and a method for producing a polymer using the ribosomal LSU disclosed below.

[0013] Item 1-1. A method for producing a ribosome LSU in a cell-free protein synthesis system, the method comprising: assembling LSU r-protein and LSU rRNA in a cell-free protein synthesis system to form a ribosome LSU, In the cell-free protein synthesis system, at least one of the LSU r-proteins is produced from a template nucleic acid encoding the LSU r-protein.

[0014] Item 1-2. The production method according to Item 1-1, wherein at least one of the LSU rRNAs is produced from a template DNA encoding the LSU rRNA in the cell-free protein synthesis system.

[0015] Item 1-3. The production method according to Item 1-1 or 1-2, wherein the ribosomal LSU is derived from Escherichia coli.

[0016] Item 1-4. The production method according to Item 1-3, wherein the ribosomal large subunit protein constituting the ribosomal LSU does not contain 50S r-protein L11 but contains 32 or less 50S r-proteins.

[0017] Item 1-5. The production method according to any one of Items 1-1 to 1-4, wherein the LSU rRNA is subjected to a mutation that imparts drug resistance.

[0018] Item 1-6. The production method according to Item 1-5, wherein the ribosomal LSU is derived from Escherichia coli, and the 23S rRNA is mutated to A2058U.

[0019] Item 1-7. The production method according to any one of Items 1-1 to 1-6, wherein all of the LSU r-protein to be assembled is produced from a template nucleic acid encoding the LSU r-protein in the cell-free protein synthesis system.

[0020] Item 1-8. The production method according to any one of Items 1-1 to 1-7, wherein the concentration of the template nucleic acid encoding the LSU r-protein in the cell-free protein synthesis system is 1 fM to 1 μ M.

[0021] Item 1-9. The production method according to Item 1-2, wherein the concentration of the template DNA encoding LSU rRNA in the cell-free protein synthesis system is 1 fM to 1 μ M.

[0022] Item 1-10. The production method according to any one of Items 1-1 to 1-9, wherein the cell-free protein synthesis system contains 1 pM to 1 M of ribosomes for translating mRNA transcribed from a template DNA encoding the LSU r-protein.

[0023] Item 1-11. The production method according to any one of Items 1-1 to 1-10, wherein the cell-free protein synthesis system comprises polysucrose.

[0024] Item 1-12. The production method according to Item 1-11, wherein the cell-free protein synthesis system contains 0.1 to 20 wt / v% of polysucrose.

[0025] Item 1-13. A method for producing a polymer in a cell-free synthesis system, comprising the following steps: A first step, wherein LSU r-protein and LSU rRNA are assembled in a cell-free protein synthesis system to produce ribosome LSU; and A second step, wherein a polymer is synthesized from a template nucleic acid encoding a sequence of the polymer in a cell-free synthesis system containing a ribosome having the ribosomal LSU obtained in the first step; In the cell-free protein synthesis system of the first step, at least one of the LSU r-proteins is produced from a template nucleic acid encoding the LSU r-protein in the cell-free protein synthesis system.

[0026] In another embodiment of the present invention, there are provided a method for producing a ribosomal SSU and a method for producing a polymer using the ribosomal SSU disclosed below.

[0027] Item 2-1. A method for producing a ribosomal SSU in a cell-free protein synthesis system, the method comprising: assembling an SSU r-protein and an SSU rRNA in a cell-free protein synthesis system to form a ribosomal SSU, In the cell-free protein synthesis system, at least two of the SSU r-proteins are produced from a template nucleic acid encoding the SSU r-protein.

[0028] Item 2-2. The production method according to Item 2-1, wherein the SSU rRNA is produced from a template DNA encoding SSU rRNA in the cell-free protein synthesis system.

[0029] Item 2-3. The production method according to Item 2-1 or 2-2, wherein the ribosomal SSU is derived from Escherichia coli.

[0030] Item 2-4. The production method according to any one of Items 2-1 to 2-3, wherein the anti-Shine-Dalgarno sequence of the SSU rRNA is set to be capable of recognizing a Shine-Dalgarno sequence that is not recognized by the anti-Shine-Dalgarno sequence of the SSU rRNA of the ribosome used in translation in the cell-free protein synthesis system.

[0031] Item 2-5. The production method according to any one of Items 2-1 to 2-4, wherein the SSU rRNA is subjected to a mutation that imparts drug resistance.

[0032] Item 2-6. The production method according to Item 2-5, wherein the ribosomal SSU is derived from Escherichia coli, and the 16S rRNA is mutated to C1192U.

[0033] Item 2-7. The production method according to any one of Items 2-1 to 2-6, wherein at least one of the SSU r-proteins is subjected to a mutation that imparts drug resistance.

[0034] Item 2-8. The production method according to Item 2-7, wherein the ribosomal SSU is derived from Escherichia coli, and the 30Sr-protein S12 is mutated to K43T.

[0035] Item 2-9. The production method according to any one of Items 2-1 to 2-8, wherein all of the SSU r-proteins to be assembled are produced from a template nucleic acid encoding an SSU r-protein in the cell-free protein synthesis system.

[0036] Item 2-10. The production method according to any one of Items 2-1 to 2-9, wherein the concentration of the template nucleic acid encoding the SSU r-protein in the cell-free protein synthesis system is 1 fM to 1 μ M.

[0037] Item 2-11. The production method according to Item 2-2, wherein the concentration of the template DNA encoding SSU rRNA in the cell-free protein synthesis system is 1 fM to 1 μ M.

[0038] Item 2-12. The production method according to any one of Items 2-1 to 2-11, wherein the cell-free protein synthesis system contains 1 pM to 1 M of ribosomes for translating mRNA transcribed from a template DNA encoding an SSU r-protein.

[0039] Item 2-13. The production method according to any one of Items 2-1 to 2-12, wherein the cell-free protein synthesis system comprises polysucrose.

[0040] Item 2-14. The production method according to Item 2-13, wherein the cell-free protein synthesis system contains 0.1 to 20 wt / v% of polysucrose.

[0041] Item 2-15. A method for producing a polymer in a cell-free synthesis system, comprising the following steps: A first step, wherein SSU r-protein and SSU rRNA are assembled in a cell-free protein synthesis system to produce ribosomal SSU; and The second step comprises synthesizing a polymer from a template nucleic acid encoding a sequence of the polymer in a cell-free synthesis system containing a ribosome having the ribosomal SSU obtained in the first step, In the cell-free protein synthesis system of the first step, at least two of the SSU r-proteins are produced from a template nucleic acid encoding the SSU r-protein.

[0042] In another embodiment of the present invention, there are provided a method for producing a ribosome and a method for producing a polymer using the ribosome as disclosed below.

[0043] Item 3-1. A method for producing ribosomes in a cell-free protein synthesis system, the method comprising: assembling LSU r-protein and LSU rRNA and assembling SSU r-protein and SSU rRNA in a cell-free protein synthesis system to form ribosomes, In the cell-free protein synthesis system, at least one of the LSU r-protein and the SSU r-protein is produced from a template nucleic acid encoding the protein.

[0044] Item 3-2. The production method according to Item 3-1, wherein the ribosome is derived from Escherichia coli.

[0045] Item 3-3. The production method according to Item 3-2, wherein the LSU r-protein constituting the ribosomal LSU does not include the 50S r-protein L11 but includes 32 or less 50S r-proteins.

[0046] Item 3-4. The production method according to any one of Items 3-1 to 3-3, wherein the LSU rRNA is subjected to a mutation that imparts drug resistance.

[0047] Item 3-5. The production method according to Item 3-4, wherein the ribosomal LSU is derived from Escherichia coli, and the 23S ribosomal RNA is mutated to A2058U.

[0048] Item 3-6. The production method according to any one of Items 3-1 to 3-5, wherein the anti-Shine-Dalgarno sequence of the SSU rRNA is set to be able to recognize a Shine-Dalgarno sequence that is not recognized by the anti-Shine-Dalgarno sequence of the SSU rRNA of the ribosome used in translation in the cell-free protein synthesis system.

[0049] Item 3-7. The production method according to any one of Items 3-1 to 3-6, wherein the SSU rRNA is subjected to a mutation that imparts drug resistance.

[0050] Item 3-8. The production method according to Item 3-7, wherein the ribosomal SSU is derived from Escherichia coli, and the 16S rRNA is mutated to C1192U.

[0051] Item 3-9. The production method according to any one of Items 3-1 to 3-8, wherein at least one of the SSU r-proteins is subjected to a mutation that imparts drug resistance.

[0052] Item 3-10. The production method according to Item 3-9, wherein the ribosomal SSU is derived from Escherichia coli, and the 30Sr-protein S12 is mutated to K43T.

[0053] Item 3-11. The production method according to any one of Items 3-1 to 3-10, wherein in the cell-free protein synthesis system, all LSU r-proteins to be assembled are produced from a template nucleic acid encoding an LSU r-protein.

[0054] Item 3-12. The production method according to any one of Items 3-1 to 3-11, wherein all of the SSU r-proteins to be assembled are produced from a template nucleic acid encoding an SSU r-protein in the cell-free protein synthesis system.

[0055] Item 3-13. The production method according to any one of Items 3-1 to 3-12, wherein at least one of LSU rRNA and SSU rRNA is produced from a template DNA encoding the RNA in the cell-free protein synthesis system.

[0056] Item 3-14. The production method according to any one of Items 3-1 to 3-13, wherein the concentration of the template nucleic acid encoding the r-protein in the cell-free protein synthesis system is 1 fM to 1 μ M.

[0057] Item 3-15. The production method according to Item 3-13, wherein the concentration of the template DNA encoding rRNA in the cell-free protein synthesis system is 1 fM to 1 μ M.

[0058] Item 3-16. The production method according to any one of Items 3-1 to 3-15, wherein the cell-free protein synthesis system contains 1 pM to 1 M of ribosomes for translating mRNA transcribed from a template DNA encoding an r-protein.

[0059] Item 3-17. The production method according to any one of Items 3-1 to 3-16, wherein the cell-free protein synthesis system comprises polysucrose.

[0060] Item 3-18. The production method according to Item 3-17, wherein the cell-free protein synthesis system contains 0.1 to 20 wt / v% of polysucrose.

[0061] Item 3-19. A method for producing a polymer in a cell-free synthesis system, comprising the following steps: A first step, wherein the assembly of LSU r-protein and LSU rRNA and the assembly of SSU r-protein and SSU rRNA are performed in a cell-free protein synthesis system to form ribosomes; and The second step comprises synthesizing a polymer from a template nucleic acid encoding a sequence of the polymer in a cell-free synthesis system containing the ribosomes obtained in the first step, In the cell-free protein synthesis system of the first step, at least one of the LSU r-protein and the SSU r-protein is produced from a template nucleic acid encoding the protein.

[0062] Effects of the Invention According to one embodiment of the production method of the present invention, a ribosome LSU containing at least one LSU r-protein produced using a template nucleic acid can be formed by a one-pot reaction in a cell-free protein synthesis system. In addition, according to another embodiment of the production method of the present invention, a ribosome SSU containing at least two SSU r-proteins produced using a template nucleic acid can be formed by a one-pot reaction in a cell-free protein synthesis system. Furthermore, according to another embodiment of the production method of the present invention, a ribosome containing at least one r-protein produced using a template nucleic acid can be formed by a one-pot reaction in a cell-free protein synthesis system.

[0063] According to the production method of the present invention, one or more or two or more r-proteins are generated from a template nucleic acid to form a ribosome LSU, a ribosome SSU or a ribosome, so by mutating the template nucleic acid, it is easy to introduce mutations into the r-protein. In addition, according to the production method of the present invention, in the cell-free protein synthesis system that forms the ribosome LSU, the ribosome SSU or the ribosome, a template DNA encoding rRNA can also be used as a supply source of rRNA, so by mutating the template DNA, it is also possible to easily introduce mutations into rRNA. In this way, according to the production method of the present invention, mutations can be freely introduced into r-proteins and rRNA, so ribosome LSUs, ribosome SSUs or ribosomes having various catalytic activities can be developed. In addition, by providing a ribosome LSU, ribosome SSU or ribosome in which the catalytic activity is changed using the production method of the present invention, it is also possible to accelerate the development of technologies such as the improvement of the translation efficiency of peptides and proteins, the improvement of the translation efficiency of peptides and proteins whose translation efficiency is low in natural ribosomes, the synthesis of peptides and proteins containing non-natural amino acids, the synthesis of peptides and proteins containing non-natural monomers other than non-natural amino acids, and the synthesis of non-natural polymers having bonds other than peptide bonds such as ester bonds and thioester bonds.

[0064] In addition, in recent years, non-natural polymers containing non-natural monomers such as non-natural amino acids, non-natural polymers having bonds other than peptide bonds, etc. have attracted attention as pharmaceutical compounds, and the development of these pharmaceutical compounds is also promoted by providing ribosomal LSU, ribosomal SSU or ribosomes in which the catalytic activity is changed using the production method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 (a) is the experimental scheme for in vitro ribosomal SSU formation. Figure 1 (b) is a graph showing the results of femtoliter droplet assay in which the conditions of the first reaction were set to 20 nM of native ribosomes (endogenous ribosomes), 0.25 nM of each of 21 plasmids containing 21 SSU r-protein genes, and 1 nM of a plasmid containing an rRNA operon encoding 16S rRNA with or1-oASD and C1192U spectinomycin resistance (SpcR), and SSU formation was performed. Figure 1 In (b), the scale bar is 10 μ m. Figure 1 (c) shows the results of exploring the concentrations of native ribosomes, a plasmid containing the SSU r-protein gene, and a plasmid containing the rRNA operon with or1-oASD and SpcR in the first reaction using a simplex-lattice design. Figure 1 In (c), RFU is the average of the relative fluorescence of the fluorescent droplets. Figure 1 (d) is the result of further exploring the optimal concentrations of native ribosomes, plasmids containing SSUr-protein genes, and plasmids containing rRNA operons having or1-oASD and SpcR in the first reaction. Figure 1 In (d), RFU is the average of the relative fluorescence of the fluorescent droplets. Figure 1 (e) is a representative micrograph of droplets when SSU was formed under optimized conditions and subjected to femtoliter droplet analysis. Figure 1 In (e), the scale bar is 10 μ m. Figure 1 (f) shows the results of bulk assay for SSU translation activity when SSU was formed under the optimized conditions (native ribosome 80 nM, 21 plasmids containing 21 SSU r-protein genes 0.05 nM each, and plasmid containing rRNA operon with or1-oASD and SpcR 0.3 nM). Figure 1 In (f), "reconstructed SSU biosynthesis" indicates the optimized conditions, "w / o native ribosomes" indicates the conditions where native ribosomes are removed from the optimized conditions, "w / o 21 SSU genes" indicates the conditions where 21 plasmids containing 21 SSU r-protein genes are removed from the optimized conditions, "w / orRNA operon" indicates the conditions where the plasmid containing the rRNA operon containing or1-oASD and SpcR is removed from the optimized conditions, and iSAT indicates the case where SSU is reconstructed by iSAT. In addition, Figure 1 In (f), RFU is relative fluorescence unit, and the data are mean ± SD (n = 3). P < 0.001, P < 0.01, ns means no significant difference (Dunnett's test relative to w / o native ribosomes). Figure 1 (g) is the result of using a plasmid containing a gene encoding a K43T mutant of 30S r-protein S12 that confers streptomycin resistance to ribosomes instead of a gene encoding 30S r-protein S12 in the first reaction, mixing only spectinomycin or a mixture of spectinomycin and streptomycin as an antibiotic in the second reaction to form SSU, and detecting the translation activity of SSU by batch analysis. Figure 1 In (g), RFU is relative fluorescence unit, NC is a negative control without using 21 plasmids containing 21 SSU r-protein genes, and the data are mean ± SD (n = 3). P < 0.01, ns means no significant difference (Welch's t-test). w / o means "without", w / means "with".

[0066] Figure 2 The results are the results of in vitro formation of ribosomal SSU using 21 or 20 plasmids containing 21 or 20 SSU r-protein genes, respectively, and a plasmid containing an rRNA operon containing or1-oASD and SpcR. Figure 2 In the table, "with 21 SSU r-protein genes" indicates the condition in which 21 plasmids containing 21 SSU r-protein genes were added, w / o S1 indicates the condition in which 20 plasmids containing 20 SSU r-protein genes other than S1 gene were added (the same applies to other S2-S21), and "w / o 21 SSU r-protein genes" indicates the condition in which 21 plasmids containing 21 SSU r-protein genes were not added. In addition, Figure 2 RFU is relative fluorescence unit.

[0067] Figure 3 (a) is the experimental scheme for in vitro ribosomal LSU formation. Figure 3 (b) shows the results of exploring the optimal concentrations of LSU formation in the first reaction, in which the native ribosome concentration was fixed at 80 nM, the concentrations of 33 plasmids containing 33 LSU r-protein genes were each in the range of 0-0.05 nM, and the concentrations of the plasmid containing the rRNA operon were in the range of 0.2-0.4 nM, wherein the rRNA operon encodes 16S rRNA with or1-oASD and SpcR and 23S rRNA with A2058U clindamycin resistance (CldR). Figure 3 In (b), RFU is the relative fluorescence unit, and w / o means “without”. Figure 3 (c) is the result of further searching for the optimal concentrations of the plasmid containing the LSU r-protein gene and the plasmid containing the rRNA operon having or1-oASD, SpcR and CldR in the first reaction. Figure 3 (d) is the result of measuring the reporter signal under the conditions optimized for the first reaction (80 nM for native ribosomes, 0.01 nM for each of 33 plasmids containing 33 LSU r-protein genes, and 0.9 nM for a plasmid containing an rRNA operon containing or1-oASD, SpcR, and CldR) by batch analysis. Figure 3In (d), "reconstructed LSU biosynthesis" indicates the optimized conditions, "w / o native ribosomes" indicates the conditions where native ribosomes are removed from the optimized conditions, "w / o 33 LSU genes" indicates the conditions where 33 plasmids containing 33 LSU r-protein genes are removed from the optimized conditions, and "w / o rRNA operon" indicates the conditions where the rRNA operon containing or1-oASD, SpcR, and CLdR is removed from the optimized conditions. Figure 3 In (d), RFU is relative fluorescence unit, and the data are mean ± SD (n = 3). P < 0.001, ns means no significant difference (Dunnett's test relative to w / o native ribosomes). Figure 3 (e) is the result of measuring the signal of LSU using pT7PCONS_EpsA20_wtSD_lacZ reporter or pT7_wtSD_LacZ reporter. "Improved reporter" is the data using pT7PCONS_EpsA20_wtSD_lacZ reporter, and "conventional reporter" is the data using pT7_wtSD_LacZ reporter. Figure 3 In (e), RFU is relative fluorescence unit, and the data are mean ± SD (n = 3). P < 0.05 (Welch's t-test).

[0068] Figure 4 The results are the results of in vitro formation of ribosomal LSU using 33 or 32 plasmids containing 33 or 32 LSU r-protein genes, respectively, and a plasmid containing an rRNA operon containing or1-oASD, SpcR, and CldR. Figure 4 In the table, "with 33 LSU r-protein genes" indicates a condition in which 33 plasmids containing 33 LSU r-protein genes were added, w / o L1 indicates a condition in which 32 plasmids containing 32 LSU r-protein genes other than L1 genes were added (the same applies to other LSU r-protein genes), and "w / o 33 LSU r-protein genes" indicates a condition in which 33 plasmids containing 33 LSU r-protein genes were not added. Figure 4 RFU is relative fluorescence unit.

[0069] Figure 5 (a) shows the results of confirming the translation activity of native ribosomes by adding native ribosomes to the first reaction and performing batch analysis in the presence or absence of thiostrepton in the second reaction in the formation of ribosomal LSU in vitro. Figure 5In (a), RFU is the relative fluorescence unit, "WT ribosome without thiostrepton" refers to the condition where thiostrepton was not added in the second reaction, and "WT ribosome with 120 nM thiostrepton" refers to the condition where thiostrepton was added in the second reaction. Figure 5 (b) shows the results of forming ribosomal LSU by performing the first reaction with or without using 32 plasmids containing 32 LSU r-protein genes except 50S r-protein L11, and measuring the signal of ribosomal LSU by batch analysis in the presence of thiostrepton in the second reaction. Figure 5 In (b), RFU is the relative fluorescence unit, "ΔL11 LSU one-pot method with 120 nM thiostrepton" refers to the condition using 32 plasmids each containing 32 LSU r-protein genes other than 50S r-protein L11, and "w / o LSU r-protein gene with 120 nM thiostrepton" refers to the condition not using 33 plasmids each containing 33 LSU r-protein genes.

[0070] Figure 6 These are the results of in vitro formation of ribosomal LSU by adding Ficoll 400 at a concentration of 0, 2, or 4.5 w / v% in the first reaction. Figure 6 In the table, “LSU one-pot w / o Ficoll” indicates the condition without the addition of Ficoll 400; “control group w / o Ficoll” indicates the condition without the use of Ficoll 400 and 33 plasmids containing 33 LSU r-protein genes (negative control); “LSU one-pot Ficoll 2%” indicates the condition with the addition of 2% Ficoll 400; “control group Ficoll 2%” indicates the condition with the addition of 2% Ficoll 400 and without the use of 33 plasmids containing 33 LSU r-protein genes (negative control); “LSU one-pot Ficoll 4.5%” indicates the condition with the addition of 4.5% Ficoll 400; “control group Ficoll 4.5%” indicates the condition with the addition of 4.5% Ficoll 400 and without the use of 33 plasmids containing 33 LSU r-protein genes (negative control). In addition, Figure 6 RFU is relative fluorescence unit.

[0071] Figure 7 The results are the result of simultaneous formation of SSU and LSU in vitro using 54 plasmids containing 54 r-protein genes (21 SSU r-protein genes and 33 LSU r-protein genes) and a plasmid containing an rRNA operon containing or1-oASD, SpcR, and CldR. Figure 7(a) is an experimental scheme for the simultaneous formation of SSU and LSU in vitro. Figure 7 (b) is the data for detecting SSU and LSU. "Reconstructed 70S ribosome biosynthesis" is the result of batch analysis of reporter signals under optimized conditions (80 nM of native ribosomes, 0.01 nM of each of 54 plasmids containing 54 r-protein genes, and 0.9 nM of a plasmid containing an rRNA operon containing or1-oASD, SpcR, and CldR). The signal of SSU was measured in the presence of spectinomycin, and the signal of LSU was measured in the presence of clindamycin. "w / o 54 ribosomal genes" means the condition where a plasmid containing 54 r-protein genes is not used. In addition, Figure 7 RFU is the relative fluorescence unit, and the data are the mean ± SD (n = 3). P < 0.01 (Welch's test).

[0072] Figure 8 The results are as follows: in the first reaction, 54 plasmids each containing 54 r-protein genes (21 SSU r-protein genes and 33 LSU r-protein genes, wherein, for the gene encoding 30S r-protein S12, a gene encoding wild-type S12 or a K43T mutant was used), and in the second reaction, batch analysis was performed in the presence of spectinomycin alone or in the presence of spectinomycin and streptomycin to confirm the formation of 70S ribosomes. Figure 8 In the table, RFU is relative fluorescence unit. “WTS12 without streptomycin” means that the gene encoding the wild-type r-protein S12 was used in the first reaction, and streptomycin was not added in the second reaction. “WT S12 with streptomycin” means that the gene encoding the wild-type r-protein S12 was used in the first reaction, and streptomycin was added in the second reaction. “S12 K43T without streptomycin” means that the gene encoding the K43T mutant of r-protein S12 was used in the first reaction, and streptomycin was not added in the second reaction. “S12 K43T with streptomycin” means that the gene encoding the K43T mutant of r-protein S12 was used in the first reaction, and streptomycin was added in the second reaction. Data are mean ± SD (n = 3).

[0073] Fig. 9 (a) is a schematic diagram of SSU and LSU with streptavidin-binding aptamer (Sb-aptamer). Fig. 9 The left figure of (b) shows that SSUs with Sb-aptamers were formed in vitro using a plasmid encoding 16S rRNA into which Sb-aptamers were inserted and 21 plasmids containing 21 SSU r-protein genes, respectively, and the translation activity of the SSUs was detected by batch analysis. Fig. 9In the left panel of (b), "w / o artificial rRNA operon" refers to a condition in which no plasmid encoding 16S rRNA was used. Fig. 9 The right figure of (b) shows that LSU with Sb-aptamer was formed in vitro using a plasmid encoding 23S rRNA into which Sb-aptamer was inserted and 33 plasmids containing 33 LSU r-protein genes, respectively, and the translation activity of the LSU was detected by batch analysis. Fig. 9 In the right figure of (b), "w / o artificial rRNA operon" refers to the condition in which no plasmid encoding 23S rRNA was used. Fig. 9 The data in (b) are mean ± SD (n = 3). P < 0.05, P < 0.01 (two-sided Welch's t-test). Fig. 9 (c) is the result of producing SSU having 16S rRNA inserted with Sb-aptamer, producing LSU having 23S rRNA inserted with Sb-aptamer, and producing and purifying them simultaneously, and analyzing the rRNA of SSU and / or LSU before and after purification by electrophoresis. Fig. 9 In (c), “Negative control w / o artificial rRNA” is the result of native ribosomes, “Artificial 16S rRNA with Sb-aptamer” is the result when SSU having 16S rRNA with Sb-aptamer inserted is produced, “Artificial 23S rRNA with Sb-aptamer” is the result when LSU having 23S rRNA with Sb-aptamer inserted is produced, and “Artificial 16S and 23S rRNA with Sb-aptamer” is the result when SSU having 16S rRNA with Sb-aptamer inserted and LSU having 23S rRNA with Sb-aptamer inserted are simultaneously produced. Fig. 9 (d) shows the result of femtoliter droplet analysis in the presence of spectinomycin and clindamycin for the newly produced artificial ribosomes SSU with Sb-aptamer and LSU with Sb-aptamer after simultaneous production and purification. Fig. 9 The left image of (d) is a representative microscopic photograph of a droplet during femtoliter droplet analysis. The scale bar is 10 μ m. Fig. 9 In the right panel of (d), RFU is relative fluorescence unit, and the data are mean ± SD (n = 3). P < 0.001 (two-sided Welch's t-test versus NC (Spc+Cld)). Fig. 9 In (d), NC (Spc+Cld) is the result when a solution obtained by adsorbing native ribosomes to streptavidin agarose and then eluting the adsorbed material (ie, foreign matter mixed in by purification on streptavidin agarose) is used.

[0074] Fig.10 (a) is a schematic diagram of four orthogonal translation systems. Fig.10 (b) is an experimental scheme for selecting orthogonal SD (oSD) sequences that do not interact with native ribosomes in E. coli cell extracts. Fig.10 (c) shows the results of measuring the reporter signal after mixing pT7_wtSD_sfGFP or pT7_oSD_sfGFP reporter with two types of Escherichia coli (E. coli) cell extracts (sonicated S12 cell extract or French press S30 cell extract). Fig.10 In (c), NC is the negative control without mixing the reporter, and the data are mean ± SD (n = 3). P < 0.01, ns means no significant difference (Dunnett's test relative to NC). Fig.10 (d) shows the result of mixing pT7_wtSD_LacZ or pT7_oSD_LacZ reporter with ultrasonically treated S12 cell extract and measuring the reporter signal. Fig.10 In (d), RFU is relative fluorescence unit, NC is negative control without reporter mixture, and data are mean ± SD (n = 3). P < 0.01, ns means no significant difference (Dunnett's test relative to NC). Fig.10 (e) is an experimental scheme for screening orthogonal anti-SD (oASD)-oSD pairs showing bilateral orthogonality in Escherichia coli (E. coli) cell extracts. Fig.10 (f) shows the result of measuring the reporter signal after mixing spectinomycin and pT7_wtSD_LacZ reporter in an E. coli cell extract containing artificial ribosomes containing ASD or oASD (b, or1, or4) and SpcR. Fig.10 In (f), RFU is the relative fluorescence unit, NC is the negative control without the reporter, and the data are the mean ± SD (n = 3). P < 0.05, ns means no significant difference (Welch's t-test). Fig.10 (g) shows the result of mixing spectinomycin and the homologous pT7_oSD_LacZ reporter with an E. coli cell extract containing artificial ribosomes containing each oASD (b, or1, or4) and SpcR, and measuring the reporter signal. Fig.10 In (g), RFU is relative fluorescence unit, NC is negative control without reporter, and data are mean ± SD (n = 3). P < 0.01, ns means no significant difference (Welch's t-test).

[0075] Fig.11 These are the results of optimizing the culture conditions for preparing the cell extract containing artificial ribosomes. Fig.11 In English, w / o means "without", and w / means "with".

[0076] Fig.12 These are the results of optimizing the spectinomycin concentration for detecting the fluorescence of the LacZ reporter. Fig.12 (a) and (b) are the results of the fluorescence intensity of the LacZ reporter obtained over time. Fig.12 (c) is the result showing the fluorescence intensity of the LacZ reporter at the end point. Fig.12 In the table, RFU is the relative fluorescence unit, NC is the negative control without the pT7_wtSD_LacZ reporter, and the data are the mean ± SD (n = 3). P < 0.05, P < 0.001, ns means no significant difference (Dunnett's test relative to NC).

[0077] Fig.13 (a) is the result of confirming the transcription and translation activity of the prepared Escherichia coli (E. coli) cell extract. Fig.13 In (a), RFU is the relative fluorescence unit, NC is the negative control without the pT7_wtSD_LacZ reporter, and the data are the mean ± SD (n = 3). P < 0.05, P < 0.01 (Welch's t-test). Fig.13 (b) shows the results of a control experiment conducted to verify the bilateral orthogonality of the or1-oASD and oSD pair. Fig.13 In (b), RFU is relative fluorescence unit, NC is negative control without LacZ reporter, and data are mean ± SD (n = 3). P < 0.05, ns means no significant difference (Dunnett's test relative to NC).

[0078] Fig.14 (a) is a schematic diagram comparing the previous batch analysis and femtoliter droplet analysis. Fig.14 (b) is a schematic diagram of automatic analysis of femtoliter droplets using deep learning. Fig.14 (c) is for the native ribosome and 4.9 μM of the sonicated S12 cell extract containing artificial ribosomes with or1-oASD and SpcR, and the control sonicated S12 cell extract containing only native ribosomes, diluted to 10 3 or 10 5 times, in pT7_oSD_or1_LacZ reporter and 100 μ The reporter signal was determined by femtoliter droplet analysis in the presence of M spectinomycin. Fig.14 In (c), RFU is relative fluorescence unit, PC is a positive control using only the sonicated S12 cell extract containing artificial ribosomes having or1-oASD and SpcR (undiluted), and NC is a negative control using only the sonicated S12 cell extract containing control. Fig.14 In the scatter plot of (c), the vertical axis is the average RFU of each droplet, and the horizontal axis is the ID of each droplet.

[0079] Fig.15 In the figure, the left image is a bright field image of a droplet in femtoliter droplet analysis, and the right image is a binary image (droplet or background) after the bright field image is converted with an accuracy of more than 90% using automatic analysis of femtoliter droplets using deep learning. In the right image, the white area represents the droplet, and the black area represents the background. Fig.15 2 examples of droplet images of femtoliter droplet analysis are shown in FIG. 1 (Example 1 and Example 2). Fig.15 The scale is 5 μ m.

[0080] Fig.16 (a) shows a control of sonicated S12 cell extract containing only native ribosomes, in which 5 nM pT7_oSD_or1_LacZ reporter and 100 μ Representative micrographs of droplets after femtoliter droplet analysis under the conditions of M spectinomycin. Fig.16 In (a), w / o means “without”, w / means “with”, and the scale is 5 μ m. Fig.16 (b) is included in 4.9 μ The cell extract containing artificial ribosomes with or1-oASD and SpcR was mixed with 5 nM pT7_oSD_or1_LacZ reporter and 100 μ Representative micrographs of droplets from femtoliter droplet analysis under the conditions of M spectinomycin. Fig.16 In (b), w / o means “without”, w / means “with”, and the scale is 5 μ m.

[0081] Fig.17For use Fig.14 The result of creating a scatter plot of the average RFU versus the diameter of each droplet for the data group (c). DETAILED DESCRIPTION

[0082] 1. Terminology Unless otherwise specified, the terms used in this specification have the meanings commonly understood by those skilled in the art in the fields of molecular biology, microbiology, organic chemistry, medicine, pharmacy, etc. If a term defined in this specification does not have the same meaning as the commonly understood meaning, the content of this specification takes precedence.

[0083] In the present invention, the "cell-free protein synthesis system" means that the transcription and translation of template DNA or the translation of template mRNA is performed in vitro to synthesize proteins without directly using cells such as Escherichia coli.

[0084] In the present invention, the "cell-free synthesis system" means that the polymer is synthesized by performing transcription and translation of template DNA or translation of template mRNA in vitro without directly using cells such as Escherichia coli.

[0085] In the present invention, "ribosome" refers to a molecular complex that reads the genetic information of mRNA and synthesizes proteins. Ribosomes are composed of two subunits, the small subunit (SSU) and the large subunit (LSU). For example, the natural ribosome of Escherichia coli, which is a prokaryotic organism, is usually a 70S ribosome that includes a 30S subunit (SSU) composed of one rRNA (16S rRNA) and 21 r-proteins, and a 50S subunit (LSU) composed of two rRNAs (5S rRNA and 23S rRNA) and 33 r-proteins. In addition, the natural ribosome of humans, which is a eukaryotic organism, is usually a 40S subunit (SSU) composed of one rRNA (18S rRNA) and 33 r-proteins, and an 80S ribosome that includes a 60S subunit (LSU) composed of three rRNAs (28S rRNA, 5.8S rRNA and 5S rRNA) and 47 r-proteins. In addition, the natural ribosome of the hyperthermophilic archaeon (Pyrococcus furiosus) which is an archaeon is generally composed of a 30S subunit (SSU) composed of one rRNA (16S rRNA) and 25 r-proteins, and a 70S ribosome composed of a 50S subunit (LSU) composed of two rRNAs (5S rRNA and 23S rRNA) and 39 r-proteins.

[0086] In the present invention, "ribosomal large subunit protein (LSU r-protein)" refers to a protein constituting LSU, and "ribosomal small subunit protein (SSU r-protein)" refers to a protein constituting SSU. In addition, in the present invention, when "ribosomal protein (r-protein)" is expressed, both LSU r-protein and SSU r-protein are included.

[0087] In the present invention, "large ribosomal subunit RNA (LSU rRNA)" refers to rRNA constituting LSU, and "small ribosomal subunit RNA (SSU rRNA)" refers to rRNA constituting SSU. In addition, in the present invention, when "ribosomal RNA (rRNA)" is expressed, both LSU rRNA and SSU rRNA are included.

[0088] In the present invention, “assembly of LSU r-protein and LSU rRNA” means that LSU r-protein and LSU rRNA associate to form ribosomal SSU, and “assembly of SSU r-protein and SSU rRNA” means that SSU r-protein and SSU rRNA associate to form ribosomal SSU.

[0089] In the present invention, the description of the number of r-proteins refers to the number of r-protein types unless otherwise specified. In addition, in the present invention, the description of the number of rRNAs refers to the number of rRNA types unless otherwise specified.

[0090] 2. Method for producing ribosomal LSU In one embodiment of the present invention, a method for producing ribosomal LSU in a cell-free protein synthesis system (hereinafter, sometimes referred to as production method 1 of the present invention) can be provided. Production method 1 of the present invention is a method for producing ribosomal LSU, comprising a step of assembling LSU r-protein and LSU rRNA in a cell-free protein synthesis system to form ribosomal LSU, wherein at least one of the LSU r-proteins is produced from a template nucleic acid encoding the LSU r-protein in the cell-free protein synthesis system. Production method 1 of the present invention is described in detail below.

[0091] [Ribosomal LSU] In the production method 1 of the present invention, the ribosomal LSU to be produced may be derived from any of prokaryotes, eukaryotes, or archaea. An example of the ribosomal LSU to be produced is ribosomal LSU derived from prokaryotes, preferably ribosomal LSU derived from Escherichia coli.

[0092] In the present invention, when the term "ribosomal LSU derived from a specific biological species" is used, it includes not only the natural ribosomal LSU composed of the LSU r-protein and LSU rRNA possessed by the specific biological species, but also its variants. For example, the ribosomal LSU derived from Escherichia coli includes the natural ribosomal LSU derived from Escherichia coli and its variants. By producing the ribosomal LSU subjected to various modifications in the production method 1 of the present invention, it is possible to provide a ribosomal LSU having a modified catalytic activity, drug resistance, etc.

[0093] In the present invention, variants of ribosomal LSU include ribosomal LSU having at least one LSU r-protein having a mutation and / or a LSU rRNA having a mutation.

[0094] In addition, it can be seen that even if the number of LSU r-proteins constituting the ribosomal LSU is one or more less than that of the natural ribosomal LSU, the ribosomal LSU can be formed. For example, the natural ribosome derived from Escherichia coli contains 33 types of LSU r-proteins (50S r-proteins L1 to L6, L9 to L25, and L27 to L36), but as shown in the Example column described later, the ribosomal LSU can be formed even if not all 33 types of LSU r-proteins are used. Therefore, in the production method 1 of the present invention, the ribosomal LSU derived from Escherichia coli composed of 33 types of LSU r-proteins or less can be formed. Therefore, in the present invention, variants of ribosomal LSU also include: a ribosomal LSU in which the number of LSU r-proteins constituting the ribosomal LSU is one or more less than that of the natural ribosomal LSU; and a ribosomal LSU having at least one mutated LSU r-protein and / or a mutated LSU rRNA, and the number of LSU r-proteins constituting the ribosomal LSU is one or more less than that of the natural ribosomal LSU.

[0095] The number of LSU r-proteins constituting the ribosomal LSU produced in the production method 1 of the present invention is not particularly limited as long as the ribosomal LSU can be formed. For example, when the number of LSU r-proteins constituting the natural ribosomal LSU is X, examples thereof include X-10 to X, preferably X-5 to X, more preferably X-4 to X, further preferably X-3 to X, X-2 to X, X-1 or X. Specifically, in the production method 1 of the present invention, when the ribosomal LSU derived from Escherichia coli is produced, 23 to 33, preferably 28 to 33, more preferably 29 to 33, further preferably 30 to 33, 31 to 33, 32 or 33 LSU r-proteins out of 33 types of LSU r-proteins (50S r-proteins L1 to L6, L9 to L25 and L27 to L36) may be used. In addition, in the ribosomal LSU derived from Escherichia coli, the 50S r-protein L21 plays an important role in forming the ribosomal LSU, so when producing the ribosomal LSU derived from Escherichia coli composed of 32 or less LSU r-proteins, it is preferable to contain at least 50S r-protein L21 as a constituent LSU r-protein. In addition, it is known that the 50S r-protein L11 contained in the ribosomal LSU derived from Escherichia coli binds to thiostrepton and inactivates the ribosomal LSU, so, for example, by producing the ribosomal LSU derived from Escherichia coli composed of 32 or less LSU r-proteins without containing the 50S r-protein L11, it is possible to provide a ribosomal LSU having resistance to thiostrepton.

[0096] In addition, in the ribosomal LSU to be produced in the production method 1 of the present invention, a mutation that imparts drug resistance to at least one of the LSU rRNAs may be introduced. In the case where drug resistance is imparted to LSU rRNA in this way, if the target protein is produced in the presence of the drug using the ribosomes (nascent ribosomes) containing the ribosome LSU produced by the production method 1 of the present invention, even in the state where endogenous ribosomes are mixed, translation by the endogenous ribosomes can be suppressed, and translation by the nascent ribosomes can be performed. For example, clindamycin resistance can be imparted by introducing the A2058U mutation into the 23S rRNA derived from Escherichia coli, so when the ribosomal LSU containing the 23S rRNA having A2058U is produced by the production method 1 of the present invention, if the target protein is produced in a cell-free protein synthesis system in the presence of clindamycin using the ribosomes containing the ribosome LSU, even in the state where endogenous ribosomes are mixed, translation by the endogenous ribosomes can be suppressed.

[0097] [Source of LSU r-protein used in the formation of ribosomal LSU] In the production method 1 of the present invention, at least one of the LSU r-proteins constituting the ribosomal LSU is supplied from a template nucleic acid encoding the LSU r-protein. When the number of LSU r-proteins supplied from the template nucleic acid does not satisfy the total number of LSU r-proteins constituting the ribosomal LSU, an LSU r-protein (in the form of protein) is used as a supply source of the LSU r-protein in addition to the template nucleic acid.

[0098] That is, when all LSU r-proteins contained in ribosomal LSU are composed of LSU r-proteins produced from template nucleic acids, only template nucleic acids can be added to the cell-free protein synthesis system without adding LSU r-proteins (in the form of proteins) as a supply source of LSU r-proteins.

[0099] In addition, when a part of the LSU r-protein contained in the ribosomal LSU is composed of the LSU r-protein produced from the template nucleic acid, the remaining LSU r-protein contained in the ribosomal LSU is composed of the r-protein supplied in the form of protein. That is, in this case, the LSU r-protein (in the form of protein) can be added to the cell-free protein synthesis system together with the template nucleic acid as a supply source of the LSU r-protein.

[0100] In the production method 1 of the present invention, at least one of the LSU r-proteins constituting the ribosomal LSU may be supplied by the template nucleic acid. For example, the number of LSU r-proteins supplied by the template nucleic acid (the number of template nucleic acids used) in the LSU r-proteins constituting the ribosomal LSU may be 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, or 30 or more. As a preferred embodiment of the production method 1 of the present invention, all the LSU r-proteins constituting the ribosomal LSU are supplied by the template nucleic acid. In addition, for example, in the case of producing the ribosomal LSU derived from Escherichia coli, the number of LSU r-proteins supplied by the template nucleic acid (the number of template nucleic acids used) in the LSU r-proteins constituting the ribosomal LSU may be 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, or 32 or more. In a preferred example when producing ribosomal LSU derived from Escherichia coli , all of the LSU r-protein constituting the ribosomal LSU is supplied from the template nucleic acid.

[0101] Hereinafter, a template nucleic acid encoding an LSU r-protein used as a supply source of the LSU r-protein constituting the ribosome LSU and an LSU r-protein (protein form) used as the supply source as needed will be described.

[0102] Template nucleic acid encoding LSUr-protein The template nucleic acid encoding the LSU r-protein may be either DNA or mRNA as long as the LSU r-protein can be produced in the cell-free protein synthesis system. When the template nucleic acid is DNA, transcription and translation are performed by the cell-free protein synthesis system to provide the LSU r-protein. In addition, when the template nucleic acid is mRNA, translation is performed by the cell-free protein synthesis system to provide the LSU r-protein.

[0103] As for the template DNA encoding LSU r-protein, it is sufficient as long as the cDNA encoding LSU r-protein is connected to the promoter in an operable manner. The promoter can be any promoter suitable for the RNA polymerase used in the cell-free protein synthesis system, for example, T7 promoter, SP6 promoter, T3 promoter, various promoters used endogenously in cells, artificially designed promoters, etc. In addition, in the template DNA encoding LSU r-protein, a terminator, polyA, 3'UTR, etc. can be included on the 3' downstream side of the cDNA encoding LSU r-protein. In addition, in the template DNA encoding LSU r-protein, an enhancer can be included on the 5' upstream side or 3' downstream side of the cDNA encoding LSU r-protein. In addition, in the case of the cell-free protein synthesis system, a Shine-Dalgarno (SD) sequence can be included between the promoter and the cDNA encoding LSU r-protein. In addition, in the template DNA encoding LSU r-protein, in order to regulate expression, an operator can be provided near the promoter.

[0104] When two or more LSU r-proteins constituting ribosomal LSU are supplied from a template DNA encoding LSU r-protein, the template nucleic acid may encode one LSU r-protein per molecule or may encode two or more LSU r-proteins per molecule.

[0105] The template DNA encoding the LSU r-protein may be any double-stranded DNA, and may be circular or linear, such as a plasmid.

[0106] The template mRNA encoding LSU r-protein includes a coding region (CDS) encoding LSU r-protein, and its 5' end is connected to a 5' untranslated region (5'UTR), and its 3' end is connected to a 3' untranslated region (3'UTR). In addition, in the template mRNA encoding LSU r-protein, a regulatory sequence such as a cap or SD sequence can be added to the 5' end of the 5'UTR, and a terminator or polyA tail can be added to the 3' end of the 3'UTR.

[0107] The base sequences of cDNA and CDS encoding LSU r-protein are known. For example, regarding cDNAs encoding LSU r-proteins derived from Escherichia coli, 50S r-protein L1 has Gene ID: 948483, 50S r-protein L2 has Gene ID: 947820, 50S r-protein L3 has Gene ID: 947817, 50S r-protein L4 has Gene ID: 947818, 50S r-protein L5 has Gene ID: 947800, 50S r-protein L6 has Gene ID: 947803, 50S r-protein L9 has Gene ID: 948720, 50S r-protein L10 has Gene ID: 948490, 50S r-protein L11 has Gene ID: 948484, 50S r-protein L12 has Gene ID: 948489, 50S r-protein L13 has Gene ID: 947828, 50S r-protein L14 has Gene ID: 947830 : 947809, 50S r-protein L15 Gene ID: 947798, 50S r-protein L16 Gene ID: 947806, 50S r-protein L17 Gene ID: 947784, 50S r-protein L18 Gene ID: 947804, 50S r-protein L19 Gene ID: 947096, 50S r-protein L20 Gene ID: 945152, 50S r-protein L21 Gene ID: 949057, 50S r-protein L22 Gene ID: 947813, 50S r-protein L23 Gene ID: 947819, 50S r-protein L24 Gene ID: 947810, 50S r-protein L25 Gene ID: 945618, 50S r-protein L27 Gene ID: 945619 The genes for 50S r-protein L31 and L32 are listed in the table below.

[0108] In the production method 1 of the present invention, when producing ribosome LSU comprising an r-protein into which a mutation is introduced, a desired mutation may be introduced into a template nucleic acid encoding the LSU r-protein.

[0109] The template nucleic acid encoding the LSU r-protein can be prepared by a known genetic engineering method.

[0110] · LSUr-protein (protein form) used as needed In the production method 1 of the present invention, the LSU r-protein (in the form of protein) used as a supply source of LSU r-protein constituting ribosomal LSU may be a protein isolated from natural ribosomes or a protein produced by genetic engineering.

[0111] [Source of LSU rRNA used in the formation of ribosomal LSU] In the production method 1 of the present invention, the supply source of LSU rRNA constituting the ribosome LSU is LSU rRNA (in the form of RNA) and / or a template DNA encoding LSU rRNA. When LSU rRNA (in the form of RNA) is used as the supply source of LSU rRNA, LSU rRNA is directly assembled into the ribosome LSU in the cell-free protein synthesis system. In addition, when a template DNA encoding LSU rRNA is used as the supply source of LSU rRNA, LSU rRNA transcribed from the template DNA is assembled into the ribosome LSU in the cell-free protein synthesis system.

[0112] For example, when producing ribosome LSU derived from Escherichia coli, 5S rRNA or a template DNA encoding 5S rRNA and 23S rRNA or a template DNA encoding 23S rRNA are used as the supply sources of LSU rRNA.

[0113] In the production method 1 of the present invention, a preferred example of a supply source of LSU rRNA constituting the ribosomal LSU is a template DNA encoding LSU rRNA. Since mutations can be easily introduced into the template DNA, when a template DNA encoding LSU rRNA is used, a ribosomal LSU containing LSU rRNA into which mutations are introduced can be easily produced.

[0114] The template DNA encoding LSU rRNA may be linked to the DNA encoding LSU rRNA via a promoter in an operable manner. The promoter may be any promoter suitable for RNA polymerase used in the cell-free protein synthesis system, for example, T7 promoter, SP6 promoter, T3 promoter, various promoters used endogenously in cells, artificially designed promoters, etc. In addition, in the template DNA encoding LSU rRNA, a terminator or the like may be included on the 3' downstream side of the base sequence encoding LSU rRNA. In addition, in the template DNA encoding LSU rRNA, an operator may be provided near the promoter in order to regulate expression.

[0115] In addition, when producing ribosome LSU derived from Escherichia coli, template DNA encoding Escherichia coli 5S rRNA and template DNA encoding 23S rRNA gene, or rrnB (an operon encoding 16S rRNA, 23S rRNA, and 5S rRNA) which is the rRNA operon of Escherichia coli, etc. can also be used as the template DNA encoding LSU rRNA.

[0116] The base sequence of DNA encoding LSU rRNA is known. For example, in the case of LSU rRNA derived from Escherichia coli, Gene ID: 948471 for DNA encoding 5S rRNA and Gene ID: 948473 for DNA encoding 23S rRNA are registered in the database provided by NIH.

[0117] The template DNA encoding LSU rRNA may be any double-stranded DNA, and may be circular or linear, such as a plasmid. The template DNA encoding LSU rRNA can be prepared by a known genetic engineering method.

[0118] In the production method 1 of the present invention, when producing ribosome LSU including LSU rRNA into which a mutation is introduced, a desired mutation may be introduced into LSU rRNA (in the form of RNA) used as a supply source and / or a template DNA encoding LSU rRNA.

[0119] [Formation of ribosomal LSU in a cell-free protein synthesis system] In the production method 1 of the present invention, a supply source of LSU r-protein (a template nucleic acid encoding LSU r-protein and, if necessary, LSU r-protein (protein form)) and a supply source of LSU rRNA (LSU rRNA (RNA form) and / or a template DNA encoding LSU rRNA) are incubated in a reaction solution of a cell-free protein synthesis system to assemble LSU r-protein and LSU rRNA to form ribosome LSU.

[0120] The formation scheme of the ribosomal LSU in the production method 1 of the present invention is as follows. The template nucleic acid encoding the LSU r-protein used as the supply source of the LSU r-protein is transcribed and translated or translated when incubated in the reaction solution of the cell-free protein synthesis system to produce the LSU r-protein. In addition, when a template DNA encoding LSU rRNA is used as the supply source of the LSU rRNA, the template DNA is transcribed when incubated in the reaction solution of the cell-free protein synthesis system to produce LSU rRNA. Then, in the reaction solution of the cell-free protein synthesis system, (i) the LSU r-protein produced from the template nucleic acid encoding the LSU r-protein, (ii) the LSU r-protein added in the form of protein as the supply source of the LSU r-protein as needed (when a part of the LSU r-protein contained in the ribosomal LSU is composed of the LSU r-protein produced from the template nucleic acid), and (iii) the LSU rRNA (added in the form of RNA) and / or the LSU rRNA produced from the template DNA encoding the LSU rRNA are assembled to form the ribosomal LSU.

[0121] In the production method 1 of the present invention, the cell-free protein synthesis system may use a cell-free protein synthesis system using a cell extract, or a recombinant cell-free protein synthesis system (PURE System) in which transcription and translation or elements required for transcription are composed of only independently purified factors. In the cell-free protein synthesis system using a cell extract, for example, a cell extract of Escherichia coli, yeast, wheat germ, rabbit reticulocyte, or the like can be used as a reaction solution.

[0122] The concentration of the template nucleic acid encoding the LSU r-protein in the reaction solution of the cell-free protein synthesis system may be appropriately adjusted to a range capable of forming ribosomal LSU, depending on the type and concentration of the source of LSU rRNA contained in the reaction solution, the concentration of ribosomes used for translation in the cell-free protein synthesis system, and the like. From the viewpoint of efficiently forming ribosomal LSU, the concentration of each template nucleic acid encoding the LSU r-protein may be 1 fM to 1 μ M, preferably 1 pM to 100 pM, more preferably 5 pM to 10 pM.

[0123] When LSU r-protein (in the form of protein) is used as a supply source of LSU r-protein (when a part of LSU r-protein contained in ribosomal LSU is composed of LSU r-protein produced from a template nucleic acid), the concentration of the LSU r-protein added to the reaction solution of the cell-free protein synthesis system may be appropriately adjusted to a range in which ribosomal LSU can be formed. For example, the concentration of each LSU r-protein added in the form of protein may be 1 pM to 1 M, preferably 1 nM to 1 mM, and more preferably 10 nM to 1 μ M.

[0124] In the cell-free protein synthesis system, when LSU rRNA (in the form of RNA) is used as a supply source of LSU rRNA, the concentration of the LSU rRNA added to the reaction solution of the cell-free protein synthesis system may be appropriately adjusted to a range in which ribosome LSU can be formed. For example, the concentration of each LSU rRNA added in the form of RNA may be 1 pM to 1 M, preferably 1 nM to 1 mM, and more preferably 10 nM to 1 μ M.

[0125] In the cell-free protein synthesis system, when a template DNA encoding LSU rRNA is used as a supply source of LSU rRNA, the concentration of the template DNA added to the reaction solution of the cell-free protein synthesis system can be appropriately adjusted to a range in which ribosome LSU can be formed. From the viewpoint of efficiently forming ribosome LSU, the concentration of the template DNA can be 1 fM to 1 μ M, preferably 1 pM to 10 nM, more preferably 200 pM to 1.5 nM.

[0126] In addition, when DNA is used as a template nucleic acid encoding LSU r-protein, and when a template DNA encoding LSU rRNA is used as a supply source of LSU rRNA, a DNA-dependent RNA polymerase for transcribing these DNAs is contained in the reaction solution of the cell-free protein synthesis system. The DNA-dependent RNA polymerase can be a polymerase that matches the promoter of the template DNA. For example, when a T7 promoter is used in the template DNA, T7 RNA polymerase can be used; when a T3 promoter is used in the template DNA, T3 RNA polymerase can be used; when an SP6 promoter is used in the template DNA, SP6 RNA polymerase can be used; when various promoters used endogenously in cells are used, natural RNA polymerases (such as E. coli RNA polymerase) can be used; in the case of artificially designed promoters, any RNA polymerase that can transcribe them can be used. In the case of a cell-free protein synthesis system using a cell extract, if the cell extract contains a DNA-dependent RNA polymerase that matches the promoter of the template DNA, it is not necessary to add a DNA-dependent RNA polymerase separately. In the case of a cell-free protein synthesis system using a cell extract, if the cell extract does not contain or contains an insufficient amount of DNA-dependent RNA polymerase that matches the promoter of the template DNA, a necessary amount of DNA-dependent RNA polymerase may be added. The concentration of the DNA-dependent RNA polymerase in the reaction solution of the cell-free protein synthesis system may be appropriately set within a range that allows transcription of the template DNA.

[0127] Furthermore, in the reaction solution of the cell-free protein synthesis system, ribosomes (hereinafter referred to as endogenous ribosomes) are included in order to translate mRNA transcribed from the template DNA encoding the LSU r-protein. In the case of a cell-free protein synthesis system using a cell extract, if endogenous ribosomes capable of translating mRNA transcribed from the template DNA encoding the LSU r-protein are included in the cell extract at an appropriate concentration, endogenous ribosomes do not need to be added separately. In addition, in the case of a cell-free protein synthesis system using a cell extract, if the endogenous ribosomes are not included or are not included in an adequate amount in the cell extract, a necessary amount of endogenous ribosomes may be added. The added ribosomes may be either natural or modified as long as they can translate the LSU r-protein. The concentration of ribosomes in the reaction solution of the cell-free protein synthesis system may be appropriately adjusted to a range capable of forming ribosome LSU, depending on the concentration of the template nucleic acid encoding the LSU r-protein contained in the reaction solution. From the viewpoint of efficiently forming ribosome LSU, the concentration may be 1 pM to 1 M, preferably 1 nM to 1 mM, and more preferably 10 nM to 1 mM. μ M is more preferably 50 nM to 500 nM.

[0128] The reaction solution of the cell-free protein synthesis system contains components such as NTP (ATP, GTP, UTP and CTP), tRNA, amino acids (20 types), and ATP regeneration factors (a combination of phosphoenolpyruvate and pyruvate kinase, a combination of creatine phosphate and creatine kinase, etc.) for transcription, translation, and ATP regeneration. Furthermore, in addition to the aforementioned components, the reaction solution of the cell-free protein synthesis system may contain coenzymes (NAD, NADH, NADP, NADPH, FAD, FADH, CoA, etc.), salts (potassium glutamate, ammonium glutamate, magnesium glutamate, potassium oxalate, etc.), folic acid (folinic acid, etc.), polyamines (spermidine, putrescine, etc.), buffers, reducing agents (DTT, TCEP, CTBA, BME, etc.), crowding agents (Ficoll 70 or 400, polyethylene glycol 3350, 6000, 8000, etc.), antimicrobial agents, etc., molecular chaperones (GroEL / ES, trigger factors, DnaKJE, etc.), other known factors that support ribosome assembly, etc., and osmotic pressure regulating factors (TMAO, urea, etc.). In the case of a cell-free protein synthesis system using a cell extract, these components may not be added separately if they are contained in a sufficient amount in the cell extract. In addition, in the case of a cell-free protein synthesis system using a cell extract, when these components are not contained or not contained in sufficient amounts in the cell extract, these components can be added in necessary amounts. In the reaction solution of the cell-free protein synthesis system, the concentration of these components can be appropriately selected within the range commonly used in the cell-free protein synthesis system. In addition, in order to improve the reaction efficiency, a continuous dialysis method can also be used.

[0129] In addition, the reaction solution of the cell-free protein synthesis system may also contain a component that promotes the formation of ribosomal LSU. As such a component, for example, polysucrose can be mentioned. Polysucrose is a neutral, hydrophilic synthetic polysaccharide rich in side chains made from sucrose, and is a substance specified as CAS number 26873-85-8. There is no particular limitation on the molecular weight of polysucrose, and for example, it can be 10,000 to 5,000,000, preferably 50,000 to 1,000,000, and more preferably 300,000 to 600,000. Polysucrose is a "Ficoll R 400" (Sigma-Aldrich), "Ficoll R PM400" (GE Healthcare), "Ficoll R"PM70" (GE Healthcare), "Ficoll 400" (Fujifilm Wako Pure Chemical Industries, Ltd.), etc. are commercially available, and these commercial products may also be used. When the reaction solution of the cell-free protein synthesis system contains Ficoll, the concentration of Ficoll in the reaction solution of the cell-free protein synthesis system may be, for example, 0.1 to 20 wt / v%, preferably 0.5 to 10 wt / v%, and more preferably 1 to 6 wt / v%.

[0130] The time for incubating the reaction solution of the cell-free protein synthesis system may be any time required for the production of LSU r-protein from the template nucleic acid encoding LSU r-protein, the production of SSU rRNA from the template DNA encoding SSU rRNA when the template DNA is used as the supply source of SSU rRNA, and the assembly of ribosome LSU, and for example, it may be about 1 to 72 hours, preferably about 2 to 48 hours, and more preferably about 3 to 12 hours.

[0131] The temperature of the reaction solution for incubating the cell-free protein synthesis system may be any temperature as long as it is a temperature that can produce the production of LSU r-protein from the template nucleic acid encoding LSU r-protein, the production of LSU rRNA from the template DNA encoding LSU rRNA when the template DNA is used as a supply source of LSU rRNA, and the assembly of ribosome LSU, and examples thereof include about 5 to 50°C, preferably about 15 to 40°C, and more preferably about 25 to 37°C.

[0132] In the reaction solution after incubation, the generated ribosomal LSU exists in a single state, or when the source is the same as or closely related to the endogenous ribosome, at least a part of the generated ribosomal LSU exists in a state of being associated with the endogenous ribosomal SSU to form a ribosome. The generated ribosomal LSU (including the state of forming a ribosome associated with the endogenous ribosomal SSU) can be purified by a well-known method such as centrifugation, chromatography, magnetic beads, electrophoresis, etc. In addition, affinity purification can also be performed by adding a tag to the template nucleic acid encoding the LSU r-protein, or when using the template DNA encoding the LSU rRNA as the supply source of the LSU rRNA, adding a tag to the template DNA. Examples of the tag include MS2 tags, FLAG tags, His tags, Strep tags, GST tags, and the like.

[0133] [Production of polymer using ribosomal LSU produced by production method 1 of the present invention] The ribosomal LSU produced by the production method 1 of the present invention can form a ribosome with a ribosomal SSU derived from the same species or a closely related species, and is used in the production of a polymer in a cell-free synthesis system. In the production of a polymer in a cell-free synthesis system, a polymer can be synthesized from a template DNA or mRNA encoding a target polymer in a cell-free synthesis system using a ribosome containing the ribosomal LSU produced by the production method 1 of the present invention. The cell-free synthesis system only needs to have components that can perform transcription and translation of the template DNA, or translation of the template mRNA, depending on the type of polymer to be produced. As an example of a cell-free synthesis system, a synthesis system having the same composition as the cell-free protein synthesis system can be cited.

[0134] The ribosome containing the ribosomal LSU produced by the production method 1 of the present invention can be used, for example, in the synthesis of peptides or proteins using natural amino acids as monomers.

[0135] In addition, in the production method 1 of the present invention, mutations can be easily introduced into the LSU r-protein and / or LSU rRNA, and ribosomal LSU having various catalytic activities can be produced. Therefore, the ribosome containing the ribosomal LSU produced by the production method 1 of the present invention can also be used in the synthesis of peptides or proteins containing non-natural amino acids, for example, using non-natural amino acids as at least one of the monomers, depending on its catalytic activity. In addition, the ribosome containing the ribosomal LSU produced by the production method 1 of the present invention can also be used in the synthesis of non-natural polymers linked by ester bonds or thioester bonds, for example, using non-natural monomers that can be linked by ester bonds or thioester bonds, depending on its catalytic activity.

[0136] In addition, by implementing the production method 1 of the present invention to produce a ribosomal LSU, a polymer can also be produced by adding a ribosomal SSU derived from the same or closely related species as needed, and then adding a template DNA or mRNA encoding a polymer, and incubating with a monomer as a raw material as needed.

[0137] 3. Method for producing ribosomal SSU In one embodiment of the present invention, a method for producing a ribosomal SSU in a cell-free protein synthesis system (hereinafter, sometimes referred to as production method 2 of the present invention) can be provided. Production method 2 of the present invention is a method for producing a ribosomal SSU, which includes a step of assembling SSU r-protein and SSU rRNA in a cell-free protein synthesis system to form a ribosomal SSU, wherein at least two of the SSU r-proteins are produced from a template nucleic acid encoding the SSU r-protein in the cell-free protein synthesis system. Production method 2 of the present invention is described in detail below.

[0138] [Ribosomal SSU] In the production method 2 of the present invention, the ribosomal SSU to be produced may be derived from any of prokaryotes, eukaryotes, or archaea. An example of the ribosomal SSU to be produced is a ribosomal SSU derived from a prokaryote, preferably a ribosomal SSU derived from Escherichia coli.

[0139] In the present invention, when a ribosomal SSU is described as being derived from a specific biological species, it includes not only a natural ribosomal SSU composed of an SSU r-protein and an SSU rRNA possessed by the specific biological species, but also variants thereof. For example, a ribosomal SSU derived from Escherichia coli includes a natural ribosomal SSU derived from Escherichia coli and variants thereof. By manufacturing a ribosomal SSU to which various modifications have been made in the manufacturing method 2 of the present invention, a ribosomal SSU having modified catalytic activity, drug resistance, etc. can be provided.

[0140] In the present invention, variants of ribosomal SSU include ribosomal SSUs having at least one SSU r-protein having a mutation and / or an SSU rRNA having a mutation.

[0141] In addition, it can be seen that even if the number of SSU r-proteins constituting the ribosomal SSU is less than that of the natural ribosomal SSU by one or more, the ribosomal SSU can be formed. For example, the natural ribosome derived from Escherichia coli contains 21 types of SSU r-proteins (30S r-proteins S1 to S21), but as shown in the first column of the Examples described later, the ribosomal SSU can be formed even if all 21 types of SSU r-proteins are not used, so the ribosomal SSU derived from Escherichia coli composed of 21 types of SSU r-proteins or less can be formed in the production method 2 of the present invention. Therefore, in the present invention, variants of the ribosomal SSU also include: a ribosomal SSU in which the number of SSU r-proteins constituting the ribosomal SSU is less than that of the natural ribosomal SSU by one or more; and a ribosomal SSU having at least one SSU r-protein with a mutation and / or a SSU rRNA with a mutation, and the number of SSU r-proteins constituting the ribosomal SSU is less than that of the natural ribosomal SSU by one or more.

[0142] The number of SSU r-proteins constituting the ribosomal SSU produced in the production method 2 of the present invention is not particularly limited as long as the ribosomal SSU can be formed. For example, if the number of SSU r-proteins constituting the natural ribosomal SSU is Y, examples thereof include Y-10 to Y, preferably Y-5 to Y, more preferably Y-4 to Y, further preferably Y-3 to Y, Y-2 to X, Y-1, or Y. Specifically, in the production method 2 of the present invention, when a ribosomal SSU derived from Escherichia coli is produced, 11 to 21, preferably 16 to 21, more preferably 17 to 21, further preferably 18 to 21, 19 to 21, 20 or 21 SSU r-proteins may be used out of 21 types of SSU r-proteins (30S r-proteins S1 to S21). In addition, in the ribosomal SSU derived from Escherichia coli, 30S r-proteins L21 and S16 play an important role in forming the ribosomal SSU. Therefore, when producing a ribosomal SSU derived from Escherichia coli composed of 20 or less SSU r-proteins, it is preferred that at least 30S r-proteins S12 and S16 are included as constituent SSU r-proteins.

[0143] In the production method 2 of the present invention, in the ribosomal SSU to be produced, the anti-Shine-Dalgarno (ASD) sequence of SSU rRNA can be set to be capable of recognizing a Shine-Dalgarno sequence (SD) sequence that is not recognized by the ASD sequence of SSU rRNA of the ribosome (endogenous ribosome) used for translation in the cell-free protein synthesis system of the production method 2 of the present invention. In this way, if it is preliminarily designed that the ribosome containing the ribosomal SSU produced by the production method 2 of the present invention (hereinafter referred to as the nascent ribosome) and the endogenous ribosome recognize different SDs, when the target polymer is produced using the nascent ribosome, by using a substance having the SD sequence that can be recognized by the nascent ribosome as the template DNA or template mRNA encoding the target polymer, even in a state where endogenous ribosomes are mixed, it is possible to suppress the translation caused by the endogenous ribosomes and more specifically detect the translation caused by the nascent ribosomes.

[0144] In addition, in the ribosomal SSU to be produced in the production method 2 of the present invention, a mutation that imparts drug resistance to SSUrRNA may be introduced. In the case where drug resistance is imparted to SSU rRNA in this way, if the target polymer is produced in the presence of the drug using the ribosome (nascent ribosome) containing the ribosomal SSU produced by the production method 2 of the present invention, even in the state where endogenous ribosomes are mixed, translation caused by endogenous ribosomes can be suppressed, and translation caused by nascent ribosomes can be carried out. For example, spectinomycin resistance can be imparted by introducing a C1192U mutation into 16S rRNA derived from Escherichia coli, so when a ribosomal SSU containing 16SrRNA having C1192U is produced by the production method 2 of the present invention, if the target polymer is produced in a cell-free synthesis system in the presence of spectinomycin using the ribosome containing the ribosomal SSU, even if endogenous ribosomes are mixed, translation caused by endogenous ribosomes can be suppressed.

[0145] Furthermore, in the ribosomal SSU to be produced in the production method 2 of the present invention, a mutation that imparts drug resistance to at least one of the SSU r-proteins may be introduced. In the case where drug resistance is imparted to the SSU r-protein in this way, if the target polymer is produced in the presence of the drug using the ribosomes (nascent ribosomes) containing the ribosomal SSU produced by the production method 2 of the present invention, even in the state where endogenous ribosomes are mixed, translation by endogenous ribosomes can be suppressed, and translation by nascent ribosomes can be performed. For example, streptomycin resistance can be imparted by introducing a K43T mutation into the 30S r-protein S12 derived from Escherichia coli, so when a ribosomal SSU containing 30S r-protein S12 having K43T is produced by the production method 2 of the present invention, if the target polymer is produced in a cell-free synthesis system in the presence of streptomycin using the ribosomes containing the ribosomal SSU, even in the state where endogenous ribosomes are mixed, translation by endogenous ribosomes can be suppressed.

[0146] [Supply source of SSU r-protein used in the formation of ribosomal SSU] In the production method 2 of the present invention, at least two of the SSU r-proteins constituting the ribosomal SSU are supplied by a template nucleic acid encoding the SSU r-protein. When the number of SSU r-proteins supplied by the template nucleic acid does not satisfy the total number of SSU r-proteins constituting the ribosomal SSU, SSU r-proteins (in the form of protein) are used as a supply source of the SSU r-protein in addition to the template nucleic acid.

[0147] That is, when all SSU r-proteins contained in the ribosomal SSU are composed of SSU r-proteins produced from the template nucleic acid, only the template nucleic acid can be added to the cell-free protein synthesis system without adding SSU r-proteins (in the form of protein) as a supply source of SSU r-proteins.

[0148] In addition, when a part of the SSU r-protein contained in the ribosomal SSU is constituted by the SSU r-protein produced from the template nucleic acid, the remaining SSU r-protein contained in the ribosomal SSU is constituted by the r-protein supplied in the form of protein. That is, in this case, the SSU r-protein (in the form of protein) may be added to the cell-free protein synthesis system together with the template nucleic acid as a supply source of the SSU r-protein.

[0149] In the production method 2 of the present invention, at least two of the SSU r-proteins constituting the ribosomal SSU may be supplied by the template nucleic acid. For example, the number of SSU r-proteins supplied by the template nucleic acid in the SSU r-proteins constituting the ribosomal SSU (the number of template nucleic acids used) may be 3 or more, 5 or more, 10 or more, 15 or more, or 20 or more. As a preferred embodiment of the production method 2 of the present invention, all the SSU r-proteins constituting the ribosomal SSU are supplied by the template nucleic acid. In addition, for example, in the case of producing a ribosomal SSU derived from Escherichia coli, the number of SSU r-proteins supplied by the template nucleic acid in the SSU r-proteins constituting the ribosomal SSU (the number of template nucleic acids used) may be 2 or more, 3 or more, 5 or more, 10 or more, 15 or more, or 20 or more. In a preferred example of producing a ribosomal SSU derived from Escherichia coli, all the SSU r-proteins constituting the ribosomal SSU are supplied by the template nucleic acid.

[0150] Hereinafter, a template nucleic acid encoding an SSU r-protein used as a supply source of the SSU r-protein constituting the ribosomal SSU and an SSU r-protein (protein form) used as the supply source as needed will be described.

[0151] Template nucleic acid encoding SSUr-protein The template nucleic acid encoding the SSU r-protein may be either DNA or mRNA as long as it can produce the SSU r-protein in the cell-free protein synthesis system. When the template nucleic acid is DNA, transcription and translation are performed by the cell-free protein synthesis system to provide the SSU r-protein. In addition, when the template nucleic acid is mRNA, translation is performed by the cell-free protein synthesis system to provide the SSU r-protein.

[0152] The template DNA encoding the SSU r-protein may be connected to the cDNA encoding the SSU r-protein via a promoter in an operable manner. The structure of the template DNA encoding the SSU r-protein is the same as that of the template DNA encoding the LSU r-protein described in the column "2. Method for producing ribosomal LSU" except for the cDNA encoding the SSU r-protein.

[0153] Furthermore, the template nucleic acid encoding the SSU r-protein may encode one SSU r-protein per molecule, or may encode two or more SSU r-proteins per molecule.

[0154] The template mRNA encoding the SSU r-protein includes a coding region (CDS) encoding the SSU r-protein, and its 5' end is connected to a 5' untranslated region (5'UTR), and its 3' end is connected to a 3' untranslated region (3'UTR). The structure of the template mRNA encoding the SSU r-protein is the same as that of the template mRNA encoding the LSU r-protein described in the above "2. Method for producing ribosomal LSU" except for the CDS encoding the SSU r-protein.

[0155] The base sequences of cDNA and CDS encoding SSU r-protein are known. For example, regarding each cDNA encoding the SSU r-protein derived from Escherichia coli, Gene ID: 945536 for 30S r-protein S1, Gene ID: 947874 for 30S r-protein S2, Gene ID: 947814 for 30S r-protein S3, Gene ID: 947793 for 30S r-protein S4, Gene ID: 947795 for 30S r-protein S5, Gene ID: 948723 for 30S r-protein S6, Gene ID: 947846 for 30S r-protein S7, Gene ID: 947802 for 30S r-protein S8, Gene ID: 949000 for 30S r-protein S9, Gene ID: 947816 for 30S r-protein S10, Gene ID: 947792 for 30S r-protein S11, and Gene ID: 947793 for 30S r-protein S12. 30S r-protein S13 Gene ID: 947791, 30S r-protein S14 Gene ID: 947801, 30S r-protein S15 Gene ID: 947686, 30S r-protein S16 Gene ID: 947103, 30S r-protein S17 Gene ID: 947808, 30S r-protein S18 Gene ID: 948721, 30S r-protein S19 Gene ID: 947811, 30S r-protein S20 Gene ID: 944759, 30S r-protein S21 Gene ID: 947577 have been registered in the database provided by NIH.

[0156] In the production method 2 of the present invention, when producing a ribosomal SSU comprising an r-protein into which a mutation is introduced, a desired mutation may be introduced into a template nucleic acid encoding the SSU r-protein.

[0157] The template nucleic acid encoding the SSU r-protein can be prepared by a known genetic engineering method.

[0158] · SSUr-protein (protein form) used as needed In the production method 2 of the present invention, the SSU r-protein (in the form of protein) used as a supply source of the SSU r-protein constituting the ribosomal SSU may be a protein isolated from a natural ribosome or a protein produced by a genetic engineering method.

[0159] [Source of SSU rRNA used in the formation of ribosomal SSU] In the production method 2 of the present invention, the supply source of SSU rRNA constituting the ribosomal SSU is SSU rRNA (in the form of RNA) and / or a template DNA encoding SSU rRNA. When SSU rRNA (in the form of RNA) is used as the supply source of SSU rRNA, SSU rRNA is directly assembled into the ribosomal SSU in the cell-free protein synthesis system. In addition, when a template DNA encoding SSU rRNA is used as the supply source of SSU rRNA, SSU rRNA transcribed from the template DNA is assembled into the ribosomal SSU in the cell-free protein synthesis system.

[0160] For example, when producing ribosomal SSU derived from Escherichia coli, 16S rRNA or a template DNA encoding 16S rRNA is used as a supply source of SSU rRNA.

[0161] In the production method 2 of the present invention, a preferred example of a supply source of SSU rRNA constituting the ribosomal SSU is a template DNA encoding SSU rRNA. Since mutations can be easily introduced into the template DNA, when a template DNA encoding SSU rRNA is used, a ribosomal SSU containing SSU rRNA into which mutations are introduced can be easily produced.

[0162] The template DNA encoding SSU rRNA may be linked to the promoter in an operable manner. The structure of the template DNA encoding SSU rRNA is the same as that of the template DNA encoding LSU rRNA described in the above section "2. Method for producing ribosomal LSU" except for the DNA encoding SSU rRNA.

[0163] When producing ribosomal SSU derived from Escherichia coli, template DNA encoding Escherichia coli 16S rRNA or rrnB (an operon encoding 16S rRNA, 23S rRNA, and 5S rRNA) which is an rRNA operon of Escherichia coli can be used as the template DNA encoding SSU rRNA.

[0164] The base sequence of DNA encoding SSU rRNA is known. For example, DNA encoding SSU rRNA (16S rRNA) derived from Escherichia coli is registered as Gene ID: 948466 in a database provided by NIH.

[0165] The template DNA encoding SSU rRNA may be any double-stranded DNA, and may be circular or linear, such as a plasmid. The template DNA encoding SSU rRNA can be prepared by a known genetic engineering method.

[0166] In the production method 2 of the present invention, when producing a ribosomal SSU comprising a mutation-introduced SSU rRNA, a desired mutation may be introduced into the SSU rRNA (in the form of RNA) used as a supply source or a template DNA encoding the SSU rRNA.

[0167] [Formation of ribosomal SSU in a cell-free protein synthesis system] In the production method 2 of the present invention, a supply source of SSU r-protein (a template nucleic acid encoding SSU r-protein and, if necessary, SSU r-protein (protein form)) and a supply source of SSU rRNA (SSU rRNA (RNA form) or a template DNA encoding SSU rRNA) are incubated in a reaction solution of a cell-free protein synthesis system to assemble SSU r-protein and SSU rRNA to form a ribosome SSU.

[0168] The formation scheme of the ribosomal SSU in the production method 2 of the present invention is as follows. The template nucleic acid encoding the SSU r-protein used as the supply source of the SSU r-protein is transcribed and translated or translated when incubated in the reaction solution of the cell-free protein synthesis system to generate the SSU r-protein. In addition, when a template DNA encoding SSU rRNA is used as the supply source of the SSU rRNA, the template DNA is transcribed when incubated in the reaction solution of the cell-free protein synthesis system to generate SSU rRNA. Then, in the reaction solution of the cell-free protein synthesis system, (i) the SSU r-protein generated from the template nucleic acid encoding the SSU r-protein, (ii) the SSU r-protein added in the form of protein as a supply source of the SSU r-protein as needed (when a part of the SSU r-protein contained in the ribosomal SSU is composed of the SSU r-protein generated from the template nucleic acid), and (iii) the SSU rRNA generated from the SSU rRNA (added in the form of RNA) or the template DNA encoding the SSU rRNA are assembled to form the ribosomal SSU.

[0169] In the production method 2 of the present invention, the reaction conditions, added components, etc. in the cell-free protein synthesis system are the same as those in "2. Production method of ribosomal LSU" except that the supply source of SSU r-protein and SSU rRNA is used instead of the supply source of LSU r-protein and LSU rRNA.

[0170] The concentration of the template nucleic acid encoding the SSU r-protein in the reaction solution of the cell-free protein synthesis system may be appropriately adjusted to a range capable of forming the ribosomal SSU, depending on the type and concentration of the source of the SSU rRNA contained in the reaction solution, the concentration of the ribosomes used for translation in the cell-free protein synthesis system, and the like. From the viewpoint of efficiently forming the ribosomal SSU, the concentration of each template nucleic acid encoding the SSU r-protein may be 1 fM to 1 μ M, preferably 5pM to 1nM, more preferably 25pM to 100pM.

[0171] When SSU r-protein (in the form of protein) is used as a supply source of SSU r-protein (when a part of SSU r-protein contained in the ribosomal SSU is composed of SSU r-protein produced from a template nucleic acid), the concentration of the SSU r-protein added to the reaction solution of the cell-free protein synthesis system may be appropriately adjusted to a range in which the ribosomal SSU can be formed. For example, the concentration of each SSU r-protein added in the form of protein may be 1 pM to 1 M, preferably 1 nM to 1 mM, and more preferably 10 nM to 1 μ M.

[0172] In the cell-free protein synthesis system, when SSU rRNA (in the form of RNA) is used as a source of SSU rRNA, the concentration of the SSU rRNA added to the reaction solution of the cell-free protein synthesis system may be appropriately adjusted to a range in which ribosomal SSU can be formed. For example, the concentration of the SSU rRNA added in the form of RNA may be 1 pM to 1 M, preferably 1 nM to 1 mM, and more preferably 10 nM to 1 μ M.

[0173] In the cell-free protein synthesis system, when a template DNA encoding SSU rRNA is used as a supply source of SSU rRNA, the concentration of the template DNA added to the reaction solution of the cell-free protein synthesis system can be appropriately adjusted to a range in which ribosomal SSU can be formed. From the viewpoint of efficiently forming ribosomal SSU, the concentration of the template DNA can be 1 fM to 1 μ M, preferably 1 pM to 10 nM, more preferably 300 pM to 1 nM.

[0174] The time and temperature for incubating the reaction solution of the cell-free protein synthesis system are the same as those in the above-mentioned "2. Method for producing ribosomal LSU".

[0175] In the reaction solution after incubation, the generated ribosomal SSU exists in a single state, or when the source is the same as the endogenous ribosome or a closely related species, at least a part of the generated ribosomal SSU exists in a state of being associated with the endogenous ribosomal LSU to form a ribosome. The generated ribosomal SSU (including the state of forming a ribosome associated with the endogenous ribosomal LSU) can be purified by well-known methods such as centrifugation, chromatography, magnetic beads, electrophoresis, etc. In addition, affinity purification can also be performed by adding a tag to the template nucleic acid encoding the SSU r-protein, or when using the template DNA encoding the SSU rRNA as the supply source of the SSU rRNA, adding a tag to the template DNA. Examples of the tag include MS2 tags, FLAG tags, His tags, Strep tags, GST tags, and the like.

[0176] [Production of polymer using ribosomal SSU produced by production method 2 of the present invention] The ribosomal SSU produced by the production method 2 of the present invention can form ribosomes with ribosomal LSUs derived from the same species or closely related species, and is used in the production of polymers in a cell-free synthesis system. In the production of polymers in a cell-free synthesis system, a polymer can be synthesized from a template DNA or mRNA encoding a target polymer in a cell-free synthesis system using a ribosome containing the ribosomal SSU produced by the production method 2 of the present invention. The cell-free synthesis system only needs to have components capable of performing transcription and translation of the template DNA, or translation of the template mRNA, depending on the type of polymer to be produced. As an example of a cell-free synthesis system, a synthesis system having the same composition as the cell-free protein synthesis system can be cited.

[0177] The ribosome including the ribosomal SSU produced by the production method 2 of the present invention can be used, for example, in the synthesis of peptides or proteins using natural amino acids as monomers.

[0178] In addition, in the production method 2 of the present invention, mutations can be easily introduced into the SSU r-protein and / or SSU rRNA, and ribosomal SSUs having various activities can be produced. Therefore, the ribosome comprising the ribosomal SSU produced by the production method 2 of the present invention can be used in the synthesis of a peptide or protein comprising a non-natural amino acid, for example, using a non-natural amino acid as at least one monomer, depending on its activity. In addition, the ribosome comprising the ribosomal SSU produced by the production method 2 of the present invention can be used in the synthesis of a non-natural polymer linked by an ester bond or a thioester bond, for example, using a non-natural monomer that can be linked by an ester bond or a thioester bond, depending on its activity.

[0179] In addition, by implementing the production method 2 of the present invention to produce a ribosomal SSU, a polymer can also be produced by adding a ribosomal LSU derived from the same or closely related species as needed, and then adding a template DNA or mRNA encoding a polymer, and incubating with a monomer as a raw material as needed.

[0180] 4. Method for producing ribosomes In another embodiment of the present invention, a method for producing ribosomes in a cell-free protein synthesis system is provided (hereinafter, sometimes referred to as production method 3 of the present invention). Production method 3 of the present invention is a method for producing ribosomes, which comprises: in a cell-free protein synthesis system, assembling LSU r-protein and LSU rRNA and assembling SSU r-protein and SSU rRNA to form ribosomes, wherein in the cell-free protein synthesis system, at least one of LSU r-protein and SSU r-protein is produced from a template nucleic acid encoding the protein. Production method 3 of the present invention is described in detail below.

[0181] [Ribosomal SSU] The ribosome to be produced in the production method 3 of the present invention may be derived from any of prokaryotes, eukaryotes, or archaea. An example of the ribosome to be produced is a ribosome derived from a prokaryote, preferably a ribosome derived from Escherichia coli.

[0182] In the present invention, when it is described as a ribosome derived from a specific biological species, it includes not only natural ribosomes composed of r-protein and rRNA possessed by the specific biological species, but also variants thereof. For example, ribosomes derived from Escherichia coli include natural ribosomes derived from Escherichia coli and variants thereof. By manufacturing ribosomes subjected to various changes in the manufacturing method 3 of the present invention, ribosomes having changed catalytic activity, drug resistance, etc. can be provided.

[0183] In the present invention, the ribosome variant includes a ribosome having at least one mutated r-protein and / or a mutated rRNA.

[0184] In addition, as described above, it is known that even if the number of LSU r-proteins constituting the ribosomal LSU is one or more less than that of the natural ribosomal LSU, the ribosomal LSU can be formed. In addition, it is known that even if the number of SSU r-proteins constituting the ribosomal SSU is one or more less than that of the natural ribosomal SSU, the ribosomal SSU can be formed. Therefore, in the present invention, the ribosome variant also includes: a ribosome in which the number of r-proteins constituting the ribosomal LSU or SSU is one or more less than that of the natural ribosome; and a ribosome having at least one mutated r-protein and / or a mutated rRNA and the number of r-proteins constituting the ribosomal LSU or SSU is one or more less than that of the natural ribosome.

[0185] The number of LSU r-proteins constituting the ribosomal LSU in the ribosomes to be produced in the production method 3 of the present invention is as described in the column "2. Production method of ribosomal LSU". In addition, the number of SSU r-proteins constituting the ribosomal SSU in the ribosomes to be produced in the production method 3 of the present invention is as described in the column "3. Production method of ribosomal SSU".

[0186] In addition, in the ribosomes to be produced in the production method 3 of the present invention, the anti-Shine-Dalgarno (ASD) sequence of SSU rRNA can be set to be capable of recognizing a Shine-Dalgarno (SD) sequence that is not recognized by the ASD sequence of SSU rRNA of the endogenous ribosomes used for translation in the cell-free protein synthesis system of the production method 3 of the present invention. In this way, if it is preliminarily designed that the ribosomes produced by the production method 3 of the present invention (hereinafter referred to as nascent ribosomes) and the endogenous ribosomes recognize different SDs, when the target polymer is produced using the nascent ribosomes, by using a substance having an SD sequence that can be recognized by the nascent ribosomes as a template DNA or template mRNA encoding the target polymer, even in a state where endogenous ribosomes are mixed, it is possible to inhibit the translation caused by the endogenous ribosomes and more specifically detect the translation caused by the nascent ribosomes.

[0187] In addition, in the ribosomes to be produced in the production method 3 of the present invention, a mutation that confers drug resistance to at least one rRNA may be implemented. In the case where drug resistance is conferred to LSU rRNA and / or SSU rRNA in this way, if the target protein is produced in the presence of the drug using the ribosomes (nascent ribosomes) produced by the production method 3 of the present invention, even in a state where endogenous ribosomes are mixed, translation by endogenous ribosomes can be suppressed and translation by nascent ribosomes can be performed. Specific examples of mutations that confer drug resistance to rRNA are described in the columns "2. Method for producing ribosome LSU" and "3. Method for producing ribosome SSU".

[0188] Furthermore, in the ribosomes to be produced in the production method 3 of the present invention, a mutation that confers drug resistance to at least one r-protein may be introduced. In the case where drug resistance is thus conferred to the r-protein, if the target protein is produced in the presence of the drug using ribosomes (nascent ribosomes) containing the ribosomal SSU produced by the production method 3 of the present invention, even in a state where endogenous ribosomes are mixed, translation by endogenous ribosomes can be suppressed and translation by nascent ribosomes can be performed. Specific examples of mutations that confer drug resistance to the r-protein are described in the column "3. Production method of ribosomal SSU".

[0189] [Source of r-protein used in ribosome formation] In the production method 3 of the present invention, at least one of the LSU r-protein and the SSU r-protein constituting the ribosome is supplied from a template nucleic acid encoding the r-protein. When the number of r-proteins supplied from the template nucleic acid does not satisfy the total number of r-proteins constituting the ribosome, r-proteins (in the form of proteins) are used as a supply source of r-proteins in addition to the template nucleic acid.

[0190] That is, when the LSU r-protein and SSU r-protein contained in the ribosome are all composed of r-protein produced from the template nucleic acid, only the template nucleic acid can be added to the cell-free protein synthesis system without adding r-protein (in the form of protein) as a supply source of r-protein.

[0191] In addition, when a part of the LSU r-protein and SSU r-protein contained in the ribosome is constituted by the r-protein produced from the template nucleic acid, the remaining r-protein contained in the ribosome is constituted by the r-protein supplied in the form of protein. That is, in this case, the r-protein (in the form of protein) may be added to the cell-free protein synthesis system together with the template nucleic acid as a supply source of the r-protein.

[0192] In the production method 3 of the present invention, at least one of the LSU r-protein and the SSU r-protein constituting the ribosome may be supplied from the template nucleic acid.

[0193] In one embodiment of the production method 3 of the present invention, the number of LSU r-proteins supplied from the template nucleic acid in the LSU r-protein constituting the ribosome (the number of template nucleic acids used) can be 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, or 30 or more. As a preferred embodiment of the production method 3 of the present invention, all the LSU r-proteins constituting the ribosome are supplied from the template nucleic acid. In addition, for example, in the case of producing ribosomes derived from Escherichia coli, the number of LSU r-proteins supplied from the template nucleic acid in the LSU r-protein constituting the ribosome (the number of template nucleic acids used) can be 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, or 32 or more. In a preferred example when producing ribosomes derived from Escherichia coli, all of the LSU r-protein constituting the ribosomes is supplied by the template nucleic acid.

[0194] In one embodiment of the production method 3 of the present invention, the number of SSU r-proteins supplied by the template nucleic acid in the SSU r-protein constituting the ribosome (the number of template nucleic acids used) can be 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 15 or more, or 20 or more. As a preferred embodiment of the production method 3 of the present invention, all the SSU r-proteins constituting the ribosome are supplied by the template nucleic acid. In addition, for example, in the case of producing ribosome SSU derived from Escherichia coli, the number of SSU r-proteins supplied by the template nucleic acid in the SSU r-protein constituting the ribosome (the number of template nucleic acids used) can be 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 15 or more, or 20 or more. In a preferred example when producing ribosomes derived from Escherichia coli, all the SSU r-proteins constituting the ribosome are supplied by the template nucleic acid.

[0195] The template nucleic acid encoding the r-protein used as a supply source of the r-protein constituting the ribosome, and the r-protein (protein form) used as the supply source as needed are described in the columns “2. Method for producing ribosomal LSU” and “3. Method for producing ribosomal SSU”.

[0196] [Source of rRNA used in ribosome formation] In the production method 3 of the present invention, the supply source of rRNA constituting the ribosome is rRNA (in the form of RNA) and / or template DNA encoding rRNA. When rRNA (in the form of RNA) is used as the supply source of rRNA, rRNA is directly used for the formation of ribosomes in the cell-free protein synthesis system. In addition, when template DNA encoding rRNA is used as the supply source of rRNA, rRNA transcribed from the template DNA in the cell-free protein synthesis system is used for the formation of ribosomes.

[0197] For example, when producing ribosomes derived from Escherichia coli, 5S rRNA or a template DNA encoding 5S rRNA, 23S rRNA or a template DNA encoding 23S rRNA, and 16S rRNA or a template DNA encoding 16S rRNA are used as the rRNA supply source.

[0198] In the production method 3 of the present invention, a preferred example of a supply source of rRNA constituting ribosomes is a template DNA encoding rRNA. Since mutations can be easily introduced into template DNA, when using a template DNA encoding rRNA, ribosomes containing rRNA into which mutations are introduced can be easily produced.

[0199] The sequence, structure, etc. of rRNA (RNA form) used as a supply source of rRNA and template DNA encoding rRNA are as described in the above-mentioned columns “2. Method for producing ribosomal LSU” and “3. Method for producing ribosomal SSU”.

[0200] [Formation of ribosomes in a cell-free protein synthesis system] In the production method 3 of the present invention, a supply source of SSU r-protein (a template nucleic acid encoding r-protein and, if necessary, r-protein (in the form of protein)) and a supply source of rRNA (rRNA (in the form of RNA) and / or a template DNA encoding rRNA) are incubated in a reaction solution of a cell-free protein synthesis system, thereby generating an assembly of LSU r-protein and LSU rRNA and an assembly of SSU r-protein and SSU rRNA, and further causing the ribosome LSU and the ribosome SSU to associate to form a ribosome.

[0201] The formation scheme of ribosomes in the production method 3 of the present invention is as follows. The template nucleic acid encoding the r-protein used as the supply source of the r-protein is transcribed and translated or translated when incubated in the reaction solution of the cell-free protein synthesis system to generate r-protein. In addition, when the template DNA encoding rRNA is used as the supply source of rRNA, the template DNA is transcribed when incubated in the reaction solution of the cell-free protein synthesis system to generate rRNA. Then, in the reaction solution of the cell-free protein synthesis system, (i) r-protein generated from the template nucleic acid encoding the r-protein, (ii) r-protein added in the form of protein as a supply source of r-protein as needed (when a part of the r-protein contained in the ribosome is composed of r-protein generated from the template nucleic acid), (iii) rRNA (added in the form of RNA) or rRNA generated from the template DNA encoding rRNA, the assembly of LSU r-protein and LSU rRNA and the assembly of SSU r-protein and SSU rRNA are generated to form ribosome LSU and ribosome SSU. Furthermore, the formed ribosomal LSU associates with the ribosomal SSU to generate ribosomes.

[0202] In the production method 3 of the present invention, the reaction conditions, added components, etc. in the cell-free protein synthesis system are as described in the columns "2. Production method of ribosomal LSU" and "3. Production method of ribosomal SSU", except for the use of the supply sources of r-protein (LSU r-protein and SSU r-protein) and the supply sources of rRNA (LSU rRNA and SSU rRNA). In addition, in the production method 3 of the present invention, the concentration of each supply source, the incubation time and the temperature are also as described in the columns "2. Production method of ribosomal LSU" and "3. Production method of ribosomal SSU".

[0203] The reaction solution after incubation contains the generated ribosomes. In addition, when the generated ribosomes are of the same origin as the endogenous ribosomes, sometimes at least a portion of the generated ribosome SSU is associated with the endogenous ribosome LSU to form a ribosome, or at least a portion of the generated ribosome LSU is associated with the endogenous ribosome SSU to form a ribosome. The generated ribosomes can be purified by known methods such as centrifugation, chromatography, magnetic beads, electrophoresis, etc. In addition, affinity purification can also be performed by giving a tag to the template nucleic acid encoding the r-protein, or by giving a tag to the template DNA encoding rRNA when using the template DNA as the supply source of rRNA. Examples of the tag include MS2 tags, FLAG tags, His tags, Strep tags, GST tags, etc.

[0204] [Production of polymer using ribosomes produced by production method 3 of the present invention] The ribosomes produced by the production method 3 of the present invention can be used in the production of polymers in the cell-free synthesis system. In the production of polymers in the cell-free synthesis system, the polymer can be synthesized from a template DNA or mRNA encoding a target polymer in the cell-free synthesis system using the ribosomes produced by the production method 3 of the present invention. The cell-free synthesis system only needs to have a component capable of performing transcription and translation of the template DNA or translation of the template mRNA according to the type of polymer produced. As an example of a cell-free synthesis system, a synthesis system having the same composition as the cell-free protein synthesis system can be cited.

[0205] The ribosome produced by the production method 3 of the present invention can be used, for example, in the synthesis of peptides or proteins using natural amino acids as monomers.

[0206] In addition, in the production method 3 of the present invention, mutations can be easily introduced into r-proteins and / or rRNA, and ribosomes with various catalytic activities can be produced. Therefore, the ribosomes produced by the production method 3 of the present invention can also be used in the synthesis of peptides or proteins containing non-natural amino acids, for example, using non-natural amino acids as at least one of the monomers, depending on their catalytic activity. In addition, the ribosomes produced by the production method 3 of the present invention can also be used in the synthesis of non-natural polymers connected by ester bonds or thioester bonds, for example, using non-natural monomers that can be connected by ester bonds or thioester bonds, depending on their catalytic activity.

[0207] Alternatively, a polymer can be produced by adding a template DNA or mRNA encoding a target polymer and, if necessary, a raw material monomer to a reaction solution of a cell-free protein synthesis system in which ribosomes are produced by carrying out the production method 3 of the present invention and incubating the mixture.

[0208] Example The present invention is not limited by the above-mentioned embodiments and examples. Various modifications are also included in the present invention within the scope that can be easily thought of by those skilled in the art without departing from the description of the claims. The contents of the documents and the like shown in this specification are cited in their entirety by reference.

[0209] 1. Test methods 1-1. Strains and plasmids By using Red recombinase （参考文献1） , destroy BL21 Star TM The chromosomal lacZ gene of (DE3) (Thermo Fisher Scientific, Waltham, MA, USA) was expressed. The specific method is briefly described below.

[0210] pKD46 encoding bacteriophage λ-Red recombinase was introduced into E. coli. The transformant was grown in a culture medium containing 100 μ g / ml Ampicillin R The cells were propagated in 50 mL SOC medium supplemented with 10 mM L-(+)-arabinose (Nacalai Tesque, Kyoto, Japan) and 10 mM L-(+)-arabinose (Nacalai Tesque, Kyoto, Japan). pKD13 was used as a template and a primer set (H1P1 forward primer, 5′-GAAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGTGTAGGCTGGAGCTGCTTC-3′ (SEQ ID NO. 1) and P4H2 reverse primer, 5′-TTACGCGAAATACGGGCAGACATGGCCTGCCCGGTTATTAA TTCCGGGGATCCGTCGACC-3' (SEQ ID NO: 2)), a fragment of the kanamycin resistance gene (kmr) was amplified and introduced into Escherichia coli (E. coli) by electroporation. Then, the electroporated cells were incubated in a solution containing 50% kanamycin-sulfate. μ g / ml LB agar medium, and kanamycin-resistant transformants were selected. The resulting strain was described as "BL21Star TM (DE3)lacZ::kmr".

[0211] Transformation of the FLP helper plasmid pCP20 into BL21 Star TM (DE3) lacZ::kmr, kmr gene excluded. pCP20 has a temperature-sensitive replicon. To demonstrate heat induction of FLP synthesis, the transformants were non-selectively cultured at 37°C, whereby loss of kanamycin resistance was confirmed. The resulting strain was designated "BL21 Star TM (DE3)lacZ::frt".

[0212] rrnB, which is the rRNA operon of E. coli, was inserted into pET-41a (+). α The genes encoding 54 r-proteins were cloned from the genome. r-protein S1 functions as a translation factor rather than a structural component, which can increase protein production （参考文献2～5） Therefore, in this experiment, r-protein S1 was also included as one of the r-proteins. Expression of genes encoding 54 r-proteins was improved by the consensus sequence of the T7 promoter (T7PCONS) that improves transcription and translation efficiency （参考文献6）and adenosine 20mer tandem repeats (EpsA20) （参考文献7） Mutations in genes encoding rRNA and r-protein were introduced by PCR using mutation-introducing primers. Plasmids for cell-free transcription and translation (CF-TXTL) were extracted from Escherichia coli and purified before use.

[0213] Table 1 shows the strains and plasmids used in this experiment.

[0214] [Table 1]

[0215] 1-2. Preparation of ultrasonically treated S12 cell extract Ultrasonic treatment of S12 cell extracts was performed using a previously reported method. （参考文献8） The specific method is briefly shown below.

[0216] Escherichia coli (E. coli) was cultured in 200 mL of 2×YPTG medium at 37°C and pelleted by centrifugation. The cell pellet was resuspended in Buffer A (Tris 20mM, NH4Cl 100mM, MgCl210mM, EDTA·2K0.5mM, DTT 2mM, adjusted to pH=7.2 with HCl) and the suspended cells were disrupted using a Q125 Sonicator (Qsonica, Newtwn, CT, USA) at a frequency input of 20kHz and an amplitude of 50%. The energy input of ultrasonic irradiation was set to 500J relative to 1mL of cell suspension. The cell extract was centrifuged at 4°C and 12000g for 10 minutes, and the supernatant was recovered. The obtained cell extract was flash frozen with liquid nitrogen and stored at -80°C until use.

[0217] 1-3. Preparation of French press cell extract The French press S30 cell extract was used to （参考文献9和10）Prepared by applying corrections. The specific method is briefly shown below. After culturing Escherichia coli with 200 mL of 2×YPTG medium at 37°C, pelletize by centrifugation. Resuspend the cell pellet in Buffer A, and add Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) and RNase Inhibitor (QIAGEN). Next, use an EmulsiFlex-C5 homogenizer (Avestin, Ottawa, Canada) to perform a single membrane pass at a pressure of 20,000 psi to break the cells. Further add RNase inhibitor and dithiothreitol (DDT), and repeat the centrifugation twice at 4°C and 30,000g for 30 minutes. The recovered supernatant was dialyzed four times with iSAT buffer (HEPES 50 mM, Mg Glutamate 10 mM, K Glutamate 200 mM, DTT 2 mM, Spermidine 1 mM, Putrescine 1 mM, adjusted to pH = 7.6 with KOH), and then concentrated by centrifugation at 4000 g using Amicon Ultra-15 3 kDa cutoff (Merck Millipore, Burlington, MA, USA). The obtained cell extract was flash frozen with liquid nitrogen and stored at -80°C until use.

[0218] The French press S150 cell extract was used to （参考文献9和10） The specific method is briefly described below. BL21 Star carrying pT7_wtASD_rRNA TM (DE3)lacZ::frt added 50 μ 1 L of 2×YPTG medium containing 1 g / mL kanamycin was cultured at 37°C until OD600 reached 0.5. 0.1 mM isopropyl- β -D-thiogalactopyranoside (IPTG, Nacalai Tesque) was incubated, and then the cells were broken by a single pass through the membrane at a pressure of 20,000 psi using an EmulsiFlex-C5 homogenizer (Avestin), and centrifuged at 30,000 g for 30 minutes at 4°C. The recovered supernatant was centrifuged at 90,000 g at 4°C for 20 hours. The recovered supernatant was further centrifuged at 150,000 g at 4°C for 3 hours. Then, the recovered supernatant was dialyzed 4 times with iSAT buffer and concentrated using Amicon Ultra-15 3kDa cutoff (Merck Millipore, Burlington, MA, USA). The obtained cell extract was flash frozen with liquid nitrogen and stored at -80°C until use.

[0219] 1-4. Preparation of cell extract containing ribosomes having artificial rRNA Introduce the plasmid containing the artificial rRNA operon into BL21 Star TM (DE3) lacZ::frt. The resulting transformant was cultured in 2×YPTG medium at 37°C until OD600 reached 0.7. The cultured cells were incubated with 0.1 mM or 1 mM IPTG (Nacalai Tesque) for 2 or 3 hours, and cell extracts were prepared by the above method.

[0220] 1-5. Transcription and Translation in Cell-Free Lines (CF-TXTL) The test for confirming the formation of artificial ribosomes in vitro was performed through the following two-stage reaction: a first reaction for forming artificial ribosomes and a second reaction for detecting the formed artificial ribosomes.

[0221] In the first reaction, the first reaction solution 15 of the composition shown in Table 2 is μ In L, native ribosomes, a plasmid containing the r-protein gene, and a plasmid containing the rRNA operon were mixed and reacted at 37°C for 180 minutes. TM In the case of cell extracts of the DE3 strain or its derivatives, T7 RNA polymerase (T7 RNAP, New England BioLabs, Ipswich, MA, USA) was not included in the first reaction solution. In addition, in order to quantify the ribosome concentration, the （参考文献11） Similarly, 15 minutes after the start of the first reaction, kasugamycin (FUJIFILM Wako Pure Chemical Corporation) was added to the reaction solution to a concentration of 2 mM. Kasugamycin is an antibiotic isolated from Streptomyces kasugaensis, and is known to inhibit the initiation of translation by hindering the association of ribosomal subunits, but has no effect on 70S ribosomes that are in translation or stopped. （参考文献12和13） .

[0222] [Table 2]

[0223] To carry out the second reaction, prepare the cell extract solution containing 3.6 μ L, antibiotics (spectimycin 200 μ M, Streptomycin 20 μg / mL, clindamycin 3mM or thiostrepton 240nM), reporter plasmid 3nM and IPTG 1mM. In the case where the reporter plasmid expresses LacZ, the second reaction solution contains 66 μ 5-Chloromethylfluorescein di- β -D-galactopyranoside (CMFDG; Invitrogen, Waltham, MA, USA) was used as the substrate of LacZ. In the second reaction, the solution after the first reaction was 15 μ L was added with the second reaction solution 15 μ L and mixed, the signal of the reporter is measured. In the second reaction, the native ribosome is inactivated by the antibiotic contained in the second reaction solution, and the expression of the sfGFP or LacZ reporter is induced by IPTG to generate a signal of the reporter caused by the formed artificial ribosome. When detecting SSU, since an SSU containing or1-oASD is formed, more specific detection can be performed by using a reporter containing or1-oSD. The signal of the reporter is measured using a fluorescent microplate reader, Fluoroskan Ascent FL TM (Thermo Fisher Scientific) or Infinite R 200 PRO (TECAN, Mannedorf, Switzerland), quantification was performed at λex = 485 nm and λem = 535 nm.

[0224] The first reaction and the second reaction were performed using 96-well plates (polystyrene, solid bottom, half area, black-walled, Greiner Bio-One International GmbH, Kremsmunster, Austria).

[0225] Specifically, the in vitro production of ribosomal SSU is carried out according to the following steps. First, native ribosomes, 21 plasmids containing 21 SSU r-protein genes, and a plasmid containing an rRNA operon (rrnB) having or1-oASD and C1192U SpcR are mixed in a first reaction solution based on an S150 cell extract, and incubated at 37°C for 180 minutes to perform a first reaction. Next, a second reaction solution containing a plasmid encoding LacZ or sfGFP, an S150 cell extract, and, if necessary, CMFDG, spectinomycin, streptomycin, etc. is added to the solution after the first reaction for 15 minutes. μ L and mixed. The fluorescence of the reaction solution was measured by batch analysis or femtoliter droplet analysis. In batch analysis, the Fluoroskan AscentFLTM (Thermo Fisher Scientific) or Infinite R 200 PRO (TECAN), reporter signal was measured kinetically at 37°C, 120 min, 720 min or 900 min, λex = 485 nm and λem = 535 nm. The method of femtoliter droplet analysis is described later.

[0226] Specifically, the in vitro production of ribosomal LSU was carried out according to the following steps. First, native ribosomes, 33 plasmids containing 33 LSU r-protein genes, and plasmids containing or1-oASD, C1192U SpcR, and clindamycin resistance (CldR) caused by the A2058U mutation in 23S rRNA were mixed in a first reaction solution based on S150 cell extract. （参考文献25） The plasmid containing the rRNA operon (rrnB) was reacted at 37°C for 180 minutes. Then, the second reaction solution containing pT7_wtSD_LacZ or pT7PCONS_EpsA20_wtSD_LacZ, S150 cell extract, and, if necessary, CMFDG, clindamycin, thiostrepton, etc. was added to the solution after the first reaction for 15 minutes. μ The fluorescence of the reaction solution was measured by batch analysis in the same manner as in the case of SSU.

[0227] 1-6. Reconstruction of SSU based on iSAT The reconstruction of SSU based on iSAT is a reconstructed version of the reported （参考文献9和10） The method was modified by adding some modifications. Specifically, the following steps were followed. In the first reaction, all proteins of the 70S ribosome (TP70) and a plasmid containing an rRNA operon (rrnB) having or1-oASD and C1192U SpcR were mixed in a first reaction solution based on an S150 cell extract and incubated at 37°C for 180 minutes. Then, a second reaction solution containing pT7_oSD_or1_LacZ, CMFDG, spectinomycin, and an S150 cell extract was added to the solution after the first reaction for 15 minutes. μ L and mix. The fluorescence of the reaction solution is measured by batch analysis. In batch analysis, Infinite R 200 PRO (TECAN), reporter signal was measured kinetically at 37°C, 720 min, λex = 485 nm and λem = 535 nm.

[0228] 1-7. Femtoliter droplet analysis Prepare a 50 mL flask containing light mineral oil (Sigma-Aldrich Corporation, St. Louis, MO, USA), 4.5% sorbitan monooleate (Nacalai Tesque), and Triton R X-100 (Nacalai Tesque) 0.5% mixed oil. The second reaction solution was mixed with the mixed oil and tapped 20 times with a micropipette (Maruemu Corporations, Osaka, Japan) to obtain an emulsion. The obtained emulsion was incubated at 37°C for 2 to 17 hours, and bright field images and fluorescence images of the droplets were obtained over time using a confocal fluorescence microscope LSM700 (Carl Zeiss AG, Oberkochen, Germany). The 488nm laser was used with an oil immersion objective (Plan-Apochromat 40x / 1.4 Oil DIC M27, Carl Zeiss AG) and oil immersion oil (Immersol TM 518F, Carl Zeiss AG) for focusing.

[0229] 1-8. Automated Flytek Droplet Analysis Using Deep Learning Since it is difficult to accurately grasp the characteristics of droplets from bright field images, a deep learning model was designed for accurate automatic analysis. Fluorescent droplets were prepared using purified LacZ (FUJIFILM Wako Pure Chemical Corporation) and CMFDG as positive controls, and 15 bright field and fluorescent images were taken containing a total of 27,580 fluorescent droplets. （参考文献14） The pixel classification model is learned, using the positive control fluorescence image as the ground truth, and processed into a binary image (droplet or background), thereby separating the droplets from the background. A convolutional neural network architecture called U-Net is used. （参考文献15） , and built a binary model. The FastAI library was used in a virtual environment built with Anaconda (Python 3.7, torch==1.4.0+cpu, torchvision==0.5.0+cpu). （参考文献16）. The model was learned using 13 ground truth binary images and the corresponding bright field images, and the remaining 2 images were used as test data. The encoder network, Resnet34, and the load decay of 1e-2 were specified. The appropriate learning rate was retrieved using the learn.lr_find() method, and a learning rate of 1e-4 was selected. The model was learned using the fit_one_cycle() method with slice(1e-4) and pct_start=0.3 for 20 epochs. All layers were unfrozen and the learning rate was retrieved again. The entire model was learned using the fit_one_cycle() method with slice(1e-4) and pct_start=0.3 for 100 epochs. As a result, the accuracy (number of correctly classified pixels / total number of pixels) reached more than 90% in the test data. Using the learned U-Net deep learning model, the bright field image of the droplet was processed into a binary image with white and black areas representing the droplet and background, respectively. The binary image is used for particle analysis using ImageJ, and the results of the particle analysis are redirected to the fluorescent image of the same source. Using the deep learning-assisted automatic analysis pipeline, the area, average fluorescence intensity, minimum fluorescence intensity, maximum fluorescence intensity, integrated density, and centroid of each droplet can be automatically and scalably obtained objectively.

[0230] 2. Establishment of a technology for detecting the translation activity of artificial ribosomes with high precision 2-1. Development of an in vitro bilateral orthogonal translation system Ribosome synthesis is a recursive process in which existing ribosomes self-replicate to form newly formed ribosomes. Therefore, in order to investigate ribosome biogenesis, a highly specific assay for detecting the translation activity of newly formed ribosomes is useful. Translation efficiency is mainly determined by the RNA-RNA base pair formation of the SD sequence of mRNA and the anti-SD (ASD) sequence of 16S rRNA. Therefore, the generation of new SD sequences and ASD sequences can be related to the development of various orthogonal translation systems that are useful for detecting newly formed ribosomes. There are 4 types of orthogonal translation systems ( Fig.10 (a)), wherein a bilateral orthogonal translation system is required to specifically and sensitively detect the translation activity of nascent ribosomes. In bilateral orthogonality, the following three conditions need to be met. (1) mRNA with orthogonal SD (oSD) is not a substrate of native ribosomes, (2) nascent artificial ribosomes with orthogonal ASD (oASD) do not translate mRNA with wild-type SD (WT-SD), and (3) nascent artificial ribosomes with oASD efficiently translate mRNA with homologous oSD. So far, although it has been shown that some oASD-oSD pairs have bilateral orthogonality in Escherichia coli, （参考文献17） However, it is unclear whether there are oASD-oSD pairs that show bilateral orthogonality in vitro （参考文献18） .

[0231] In order to screen oASD-oSD pairs showing strong bilateral orthogonality in E. coli cell extracts, the seven orthogonal ribosome-mRNA pairs shown in Table 3 (denoted as a, b, c, d, or1, or4, and j) were selected as candidates.

[0232] [Table 3]

[0233] First, an experimental protocol was designed to select oSD sequences that do not interact with native ribosomes in E. coli cell extracts ( Fig.10 (b)). pT7_wtSD_sfGFP or pT7_oSD_sfGFP reporter was mixed with two E. coli cell extracts (sonicated S12 cell extract or French press S30 cell extract). Batch analysis results showed that 6 oSD sequences (b, c, d, or1, or4, j) did not show reporter signals and had no functional interaction or weak functional interaction with native ribosomes ( Fig.10 (c)). The results were the same between the two E. coli cell extracts. Therefore, the easy-to-prepare ultrasonic S12 cell extract was used in the following screening process. Furthermore, the orthogonality of the first four oSD sequences (b, or1, or4, j) was verified using a LacZ reporter that has a higher sensitivity than the GFP reporter. As a result, in the batch analysis, three oSD sequences (b, or1, or4) did not show reporter signals ( Fig.10 (d)).

[0234] Next, we designed an experimental scheme to screen for oASD-oSD pairs that showed bilateral orthogonality in E. coli cell extracts ( Fig.10 First, the preparation of a ribosome containing native ribosomes and oASD and C1192U spectinomycin resistance (SpcR) was studied. （参考文献19） The conditions of the E. coli cell extracts of the artificial ribosomes were as follows. TM (DE3) lacZ::frt were transformed with plasmids containing rRNA with or without C1192U spectinomycin resistance (SpcR). These strains were cultured with 0.1 mM or 1 mM IPTG for 2 or 3 h and mixed with pT7_wtSD_sfGFP reporter and with or without 10 μ The results showed that the strongest sfGFP was produced in the presence of spectinomycin under the condition of 0.1 mM IPTG treatment for 3 hours, indicating the efficient expression of artificial rRNA ( Fig.11). Therefore, in the following experiments, the condition of treating with 0.1 mM IPTG for 3 hours was adopted.

[0235] Then the optimal concentration of spectinomycin was studied. TM (DE3) lacZ::frt was transformed with plasmids containing rRNA with or without C1192U spectinomycin resistance (SpcR). These strains were cultured in the presence of 0.1 mM IPTG for 3 hours, and the sonicated S12 cell extract and pT7_wtSD_LacZ reporter were mixed at various spectinomycin concentrations. As a result, it was confirmed that at a spectinomycin concentration of 5 mM, the activity of endogenous ribosomes was strongly inhibited and the translation activity of artificial ribosomes was specifically detected ( Fig.12 Therefore, in the following experiments, unless otherwise specified, the spectinomycin concentration was set to 5 mM.

[0236] Next, the transcriptional and translational activity of the prepared E. coli cell extract was confirmed. The cell extract containing native ribosomes and artificial ribosomes with oASD (b, or1, or4) and SpcR was mixed with the pT7_wtSD_LacZ reporter. As a result, the reporter signal ( Fig.13 (a)). From this result, it can be seen that these Escherichia coli cell extracts can be functionally adjusted.

[0237] Next, we selected oASD sequences that did not interact with the pT7_wtSD_LacZ reporter. We mixed spectinomycin and the pT7_wtSD_LacZ reporter with E. coli cell extracts containing artificial ribosomes containing each oASD (b, or1, or4). As a result, no reporter signal was detected, confirming that the artificial ribosomes containing oASD (b, or1, or4) had no functional interaction or weak functional interaction with the mRNA containing WT-SD ( Fig.10 (f)). In addition, the homologous pT7_oSD_LacZ reporter and spectinomycin were mixed in the E. coli cell extract containing artificial ribosomes with each oASD (b, or1, or4), and the results confirmed that a strong reporter signal was generated in the case of the combination of artificial ribosomes with or1-oASD and SpcR and the pT7_oSD_or1_LacZ reporter ( Fig.10(g)). Furthermore, a control experiment was performed to verify the bilateral orthogonality of the or1-oASD and oSD pairs. E. coli cell extracts containing native ribosomes and artificial ribosomes with WT-ASD and SpcR were mixed with spectinomycin and pT7_wtSD_LacZ or pT7_oSD_or1_LacZ reporters. As a result, ribosomes with wtASD and SpcR did not interact with the pT7_oSD_or1_LacZ reporter ( Fig.13 (b)). That is, it was confirmed that the bilateral orthogonality of the or1-oASD and oSD pair was reliably obtained not by SpcR but by the interaction between the oASD and oSD pair. That is, from these results, it can be seen that the or1-oASD and or1-oSD pair showed strong bilateral orthogonality in the E. coli cell extract.

[0238] 2-2. Development of automated fly-droplet analysis using deep learning In order to study the optimal reaction conditions for in vitro ribosome synthesis, a high-sensitivity assay for detecting the translation activity of nascent ribosomes has become important. Therefore, a femtoliter droplet assay ( Fig.14 (a)). In this analysis, since a small amount of reaction solution is enclosed in a femtoliter droplet, the concentrations of enzyme and substrate become very high, enabling highly sensitive detection of enzyme activity. （参考文献20） Furthermore, an automated pipeline using deep learning was developed for scalable and objective analysis of femtoliter droplet analysis ( Fig.14 In the analysis pipeline, the learned U-Net is used. （参考文献15） Deep learning model, converting the bright field image of the droplet into a binary image (droplet or background) with an accuracy of more than 90% ( Fig.15 The binarized image was used for ImageJ （参考文献21） Automatic particle analysis to determine the size, fluorescence intensity, etc. of each droplet.

[0239] Next, the sensitivity of femtoliter droplet analysis was evaluated. First, artificial ribosomes containing or1-oASD and SpcR were prepared. μ M cell extracts, and control cell extracts containing only native ribosomes.

[0240] First, a control experiment was performed using a control cell extract containing only native ribosomes and the pT7_oSD_or1_LacZ reporter. 5 nM of the pT7_oSD_or1_LacZ reporter was mixed into the control cell extract. Under this condition, no fluorescence derived from native ribosomes was detected in the batch analysis ( Fig.10 (d) ), while fluorescence originating from native ribosomes was observed in femtoliter droplet analysis ( Fig.16 (a)), confirming that the femtoliter droplet analysis has high sensitivity. 5 nM pT7_oSD_or1_LacZ reporter and 100 μ The results of femtoliter droplet analysis showed that spectinomycin with 100 M can eliminate the fluorescence derived from native ribosomes.

[0241] Next, in the artificial ribosome 4.9 containing or1-oASD and SpcR μ M of cell extract, 5 nM of pT7_oSD_or1_LacZ reporter and 100 μ M of spectinomycin, and femtoliter droplet analysis confirmed that the translation activity of artificial ribosomes with or1-oASD and SpcR could be specifically detected ( Fig.16 (b)).

[0242] Therefore, next, the sensitivity of the femtoliter droplet analysis was estimated.

[0243] Sonicated S12 cell extracts containing native ribosomes and artificial ribosomes with or1-oASD and C1192U spectinomycin resistance (SpcR), and control sonicated S12 cell extracts containing native ribosomes only were diluted to 10 3 or 10 5 times, in pT7_oSD_or1_LacZ reporter and 100 μ The reporter signal was measured in femtoliter droplets in the presence of spectinomycin ( Fig.14 (c)). Since the threshold of RFU as the reporter signal is 22, it is known that 49 pM of artificial ribosomes can be detected by femtoliter droplet analysis (dilution factor 10 5 ).in addition, Fig.17 Indicates the use Fig.14 The results of creating a scatter plot of the average RFU versus the diameter of each droplet for the data group (c).

[0244] At 49 pM, the estimated diameter is 1 μ The droplets of m contain an average of 1.54×10 -2 ribosomes. According to the Poisson distribution formula, 1 μ The probability that m droplets contain k ribosomes can be expressed as follows: According to the following formula, the probability of a droplet containing 0, 1, or more than 2 ribosomes is 0.9847, 0.0152, or 0.0001, respectively.

[0245] [Number 1]

[0246] k: the number of ribosomes λ: 1.54×10 -2 At 49 pM artificial ribosomes (dilution factor 10 5 ) data, 15544 particles with diameters of 0.5 to 1.5 μ m droplets, of which 17 were fluorescent droplets. That is, the ratio of observed fluorescent droplets was 0.0011. If all were considered, it is estimated that most fluorescent droplets (89%) contained only one ribosome. From the above results, it can be seen that the translation activity of artificial ribosomes can be detected down to the single ribosome level with ultra-high sensitivity through automated analysis of femtoliter droplet analysis using deep learning.

[0247] 3. In vitro ribosome production 3-1. Production of ribosomal SSU The ribosomal SSU was produced in vitro using a bilateral orthogonal translation system and a femtoliter droplet assay. The formation and confirmation of the ribosomal SSU was performed in two reactions: Figure 1 In the first reaction, the rRNA operon (rrnB) with or1-oASD and SpcR was transcribed, 21 SSU r-protein genes were transcribed and translated, and the ribosomal SSU was assembled in the S150 cell extract to synthesize the ribosomal SSU. In the second reaction, the pT7_oSD_or1_LacZ reporter and 100 μ Spectinomycin M was used to detect the translation activity of the nascent SSU.

[0248] First, the conditions of the first reaction were set to 20 nM native ribosomes, 0.25 nM each of 21 plasmids containing 21 SSU r-protein genes, and 1 nM of a plasmid containing an rRNA operon with or1-oASD and C1192U spectinomycin resistance (SpcR). As a result, no signal representing ribosomal SSU was confirmed even in the femtoliter droplet analysis ( Figure 1 (b)). Therefore, in the simplex-lattice design, in the first reaction, the optimal concentrations were explored within the range of 0-100 nM for native ribosomes, 0-0.5 nM for 21 plasmids containing 21 SSU r-protein genes, and 0-3 nM for a plasmid containing an rRNA operon containing or1-oASD and SpcR. As a result, the reporter signal could be slightly detected by femtoliter droplet analysis ( Figure 1 (c)).

[0249] The results of the search for the optimal concentration based on the simplex grid design method suggest that reducing the concentration of the plasmid containing the SSU r-protein gene and the plasmid containing the rRNA operon is important for the reporter signal, and the expression efficiency of the structural components of the ribosome is higher than that of other genes. It is believed that this is because the synthesis of ribosomes accounts for most of the energy required for cell division, and the doubling time of ribosomes limits the doubling time of cells. Therefore, the synthesis of ribosomes is under strong selection pressure and must be highly efficient. （参考文献22和23） Therefore, the concentrations of native ribosomes, 21 plasmids containing 21 SSU r-protein genes, and plasmids containing rRNA operons containing or1-oASD and SpcR in the first reaction were further studied. Specifically, the concentrations of native ribosomes were greater than 0 nM and less than 240 nM, the concentrations of 21 plasmids containing 21 SSU r-protein genes were greater than 0 nM and less than 0.15 nM, and the concentration of plasmids containing rRNA operons containing or1-oASD and SpcR was greater than 0 nM and less than 0.9 nM. As a result, a strong reporter signal was detected ( Figure 1 (d)), in particular, under the optimal conditions of 80 nM for native ribosomes, 0.05 nM for plasmids containing 21 SSU r-protein genes, and 0.3 nM for plasmids containing rRNA operons, a reporter signal at a nearly saturated level was confirmed in the femtoliter droplet analysis ( Figure 1 In addition, under the optimized conditions, the reporter signal can be detected even in batch analysis, and the ribosomal SSU is formed more efficiently than the conventional iSAT ( Figure 1 (f)).

[0250] In addition, it was confirmed that the reporter signal originated from a newly synthesized artificial SSU composed of r-protein and rRNA from the plasmid. Specifically, the gene encoding the K43T mutant of 30S r-protein S12 that confers streptomycin resistance to ribosomes was used. （参考文献18和24） The first reaction was performed under the above-mentioned optimal conditions in place of the gene encoding the wild-type 30S r-protein S12. In the second reaction, as antibiotics, only spectinomycin or spectinomycin and streptomycin were mixed, and the reporter signal was detected by batch analysis. As a result, only when the gene encoding the 30S r-protein S12 K43T mutant was used, there was no effect on the reporter signal even in the presence of spectinomycin and streptomycin ( Figure 1 (g)). This result indicates that the nascent ribosomal SSU is composed mainly of newly synthesized rRNA and r-protein. From the above, it can be seen that the ribosomal SSU can be efficiently synthesized in vitro by simultaneously performing transcription of the rRNA operon, transcription and translation of the r-protein gene, and assembly of the SSU.

[0251] 3-2. Production of ribosomal SSU using 20 types of SSUr-protein genes SSU was produced in vitro by removing one r-protein from the 21 SSU r-proteins (30S r-proteins) that constitute SSU. In the first reaction, 80nM native ribosomes, 0.05nM of 20 plasmids containing 20 SSU r-protein genes, and 0.3nM of a plasmid containing an rRNA operon (rrnB) containing or1-oASD and SpcR were mixed at respective concentrations in a first reaction solution based on an S150 cell extract and reacted at 37°C for 180 minutes. Next, in the second reaction, the signal of ribosomal SSU was detected by batch analysis using a pT7_oSD_or1_LacZ reporter and spectinomycin.

[0252] As a result, when 30S r-protein S12 (rpsL) or 30S r-protein S16 (rpsP) was missing from the 21 SSU r-proteins, the signal of the ribosomal SSU was weak and the efficiency of ribosomal SSU formation was slightly reduced. However, even if one SSU r-protein other than 30S r-protein S12 was missing, the efficiency of ribosomal SSU formation was hardly affected ( Figure 2 ). That is, it was clarified that ribosomal SSU can be formed in vitro even without using all 21 SSU r-protein genes.

[0253] 3-3. Production of ribosomal LSU In vitro ribosomal LSU production was performed using a bilateral orthogonal translation system and batch analysis. The experimental protocol was similar to that of SSU ( Figure 3 In the first reaction, in a first reaction solution based on S150 cell extract, clindamycin resistance (CldR) caused by or1-oASD, SpcR, and A2058U mutation of 23S rRNA was detected. （参考文献25） The ribosomal LSU was synthesized by transcription of the rRNA operon (rrnB), transcription and translation of 33 LSU r-protein genes, and assembly of the ribosomal LSU. In the second reaction, the translation activity of the newly generated ribosomal LSU was detected by batch analysis using the pT7_wtSD_LacZ reporter in the presence of 1.5 mM clindamycin.

[0254] In the first reaction, the optimal concentrations were explored by fixing the native ribosome concentration at 80 nM, 0-0.05 nM for each of the 33 plasmids containing the 33 LSU r-protein genes, and 0.2-0.4 nM for the plasmid containing the rRNA operon containing or1-oASD, SpcR, and CldR. The results showed that the signal detection of ribosome LSU was the strongest under the conditions of 0.01 nM for each plasmid containing the LSU r-protein gene and 0.4 nM for the plasmid containing the rRNA operon ( Figure 3 (b)).

[0255] In addition, in the first reaction, the optimal concentrations were searched within the range of 80 nM for native ribosomes, 0-0.015 nM for each of the 33 plasmids containing 33 LSU r-protein genes, and 0.6-1.5 nM for the plasmid containing the rRNA operon. As a result, under the conditions of 80 nM for native ribosomes, 0.01 nM for each of the plasmids containing 33 LSU r-protein genes, and 0.9 nM for the plasmid containing the rRNA operon containing or1-oASD, SpcR, and CldR, the reporter signal derived from the synthetic ribosomal LSU could be strongly detected even by batch analysis ( Figure 3 (c) and Figure 3 (d)). It should be noted that the reporter signal of the nascent ribosome LSU is lower than the reporter signal of the nascent ribosome SSU ( Figure 3 (d) and Figure 1 (f)). This is believed to be because the newly formed ribosome LSU can translate both LacZ and 33 LSU r-proteins, and the ribosome with the A2058U CldR mutation only maintains less than 30% of its translation activity in the presence of clindamycin. （参考文献26） .

[0256] 3-4. Improvement of LSU signal The LacZ reporter was replaced from pT7_wtSD_LacZ to pT7PCONS_EpsA20_wtSD_lacZ with the T7CONS_EpsA20 sequence added upstream of the LacZ gene sequence, and the formation of ribosomal LSU was confirmed. Specifically, in the first reaction, 80 nM native ribosomes, 0.01 nM of 33 plasmids containing 33 LSU r-protein genes, and 0.9 nM of a plasmid containing an rRNA operon (rrnB) having or1-oASD, SpcR, and A2058U clindamycin resistance (CldR) were reacted at respective concentrations in a first reaction solution based on an S150 cell extract. Then, in a second reaction, the translation activity of the nascent LSU was detected by bulk analysis using the pT7PCONS_EpsA20_wtSD_lacZ reporter or the pT7_wtSD_LacZ reporter in the presence of 1.5 mM clindamycin.

[0257] As a result, when the pT7PCONS_EpsA20_wtSD_lacZ reporter was used, the LSU signal increased nearly 5 times compared to the case of the pT7_wtSD_LacZ reporter, and the RFU increased from 200 to 1000 ( Figure 3 (e)).

[0258] 3-5. Production of ribosomal LSU using 32 LSUr-protein genes One r-protein was removed from the 33 r-proteins (50S r-proteins) that constitute the ribosomal LSU, and LSU formation was performed in vitro. In the first reaction, 80 nM native ribosomes, 0.01 nM of 32 plasmids containing 32 LSU r-protein genes, and 0.9 nM of a plasmid containing an rRNA operon (rrnB) containing or1-oASD, SpcR, and CldR were mixed at respective concentrations in a first reaction solution based on an S150 cell extract, and reacted at 37°C for 180 minutes. Next, in the second reaction, the signal of the ribosomal LSU was detected by batch analysis using the pT7PCONS_EpsA20_wtSD_lacZ reporter and clindamycin.

[0259] As a result, when 50S r-protein L21 was missing from among the 33 LSU r-proteins, the signal of ribosomal LSU was weak and the efficiency of ribosomal LSU formation was slightly reduced. However, even if one LSU r-protein other than 50S r-protein L21 was missing, the efficiency of ribosomal LSU formation was hardly affected ( Figure 4 ). That is, it was clarified that ribosomal LSU can be formed in vitro even without using all 33 LSU r-protein genes.

[0260] 3-6. Production of LSU with Thiostrepton Resistance Thiostrepton is known to inactivate LSU by binding to 50S r-protein L11. Therefore, 50S r-protein L11 was removed from the 33 r-proteins (50S r-proteins) constituting ribosomal LSU, LSU was formed in vitro, and LSU with thiostrepton resistance was produced. In the first reaction, 80 nM native ribosomes, 0.01 nM of 32 plasmids containing 32 LSU r-protein genes except 50S r-protein L11, and 0.9 nM of a plasmid containing an rRNA operon (rrnB) containing or1-oASD, SpcR, and CldR were mixed at respective concentrations in a first reaction solution based on an S150 cell extract, and reacted at 37°C for 180 minutes. Next, in the second reaction, the translation activity of ribosomal LSU was detected by batch analysis using the pT7PCONS_EpsA20_wtSD_lacZ reporter in the presence or absence of thiostrepton.

[0261] As a result, when the activity of natural ribosomes was measured under the same conditions, the LSU signal disappeared in the presence of thiostrepton ( Figure 5 (a)), but in the case where LSU was formed using a plasmid containing 32 LSU r-protein genes other than r-protein L11, the signal of LSU was also detected in the presence of thiostrepton ( Figure 5 That is, from the present results, it was found that thiostrepton resistance could be imparted to LSU by forming ribosome LSU in vitro without using the r-protein L11 gene.

[0262] 3-7. Improvement of the efficiency of ribosome LSU formation using polysucrose The effect of Ficoll on the formation of ribosomal LSU was studied. Specifically, in the first reaction, 80 nM native ribosomes, 0.01 nM of 33 plasmids containing 33 LSU r-protein genes, 0.9 nM of a plasmid containing an rRNA operon (rrnB) containing or1-oASD, SpcR and CldR, and 0, 2 or 4.5 w / v% Ficoll were added to the first reaction solution based on S150 cell extract. R 400) were mixed at respective concentrations and reacted at 37°C for 180 minutes. Next, in the second reaction, the signal of ribosomal LSU was detected by batch analysis using pT7PCONS_EpsA20_wtSD_lacZ reporter and clindamycin.

[0263] As a result, when polysucrose was added in the first reaction to form ribosome LSU, the signal of LSU was increased compared with the case where polysucrose was not added ( Figure 6 ). That is, it was clarified that the formation efficiency of ribosome LSU was improved by adding polysucrose in the first reaction of forming ribosome LSU.

[0264] 3-8. Production of 70S ribosome (simultaneous formation of SSU and LSU) The simultaneous production of ribosomal SSU and ribosomal LSU was carried out in vitro according to the following steps. First, in the first reaction, 80 nM native ribosomes, 0.01 nM each of 21 plasmids containing 21 SSU r-protein genes, 0.01 nM each of 33 plasmids containing 33 LSU r-protein genes, and 0.9 nM of a plasmid containing an rRNA operon (rrnB) having or1-oASD, C1192U SpcR, and A2058U CldR were mixed at respective concentrations in a first reaction solution based on an S150 cell extract and reacted at 37°C for 180 minutes. Next, the reaction solution after the first reaction was divided into two parts. For one portion of the reaction solution, the signal of SSU was measured by batch analysis using pT7_oSD_or1_LacZ reporter and spectinomycin, and for another portion of the reaction solution, the signal of LSU was measured by batch analysis using pT7PCONS_EpsA20_wtSD_lacZ reporter and clindamycin.

[0265] As a result, the reporter signals of both SSU and LSU were detected, confirming that SSU and LSU can be reconstructed simultaneously in vitro ( Figure 7 (b)).

[0266] 3-9. Production of 70S ribosomes with streptomycin resistance The gene encoding the K43T mutant of 30S r-protein S12 that confers streptomycin resistance to ribosomes was used to produce 70S ribosomes having streptomycin resistance. In the first reaction, 80 nM native ribosomes, 0.01 nM plasmids containing 21 plasmids each containing 21 SSU r-protein genes (wherein, for the gene encoding 30S r-protein S12, a gene encoding wild-type S12 or a K43T mutant was used), 0.01 nM plasmids containing 33 LSU r-protein genes, and 0.9 nM plasmid containing an rRNA operon (rrnB) having or1-oASD, C1192USpcR, and A2058U CldR were mixed at respective concentrations in a first reaction solution based on an S150 cell extract and reacted at 37°C for 180 minutes. Next, in the second reaction, the translation activity of the ribosomal SSU was detected by batch analysis using the pT7_oSD_or1_LacZ reporter in the presence of spectinomycin and in the presence or absence of streptomycin.

[0267] As a result, when the wild type was used as the 30S r-protein S12 gene, no signal was detected in the presence of streptomycin, but when the gene encoding the 30S r-protein S12 K43T mutant was used, a signal was detected in the presence of streptomycin ( Figure 8 ). That is, from the present results, it was found that by using a gene encoding the 30S r-protein S12 K43T mutant as the 30S r-protein S12 gene, it was possible to produce 70S ribosomes having streptomycin resistance.

[0268] 4. In vitro Ribosome Production and Purification In this experiment, in order to directly prove that the newly generated artificial ribosomes in vitro exhibit translational activity, the newly generated artificial ribosomes were purified and their translational activity was confirmed.

[0269] 4-1. Production of SSU and LSU with streptavidin-binding aptamers First, in order to purify the newly generated artificial ribosome, plasmids encoding rRNA into which the Sb-aptamer was inserted were constructed. The three plasmids constructed were shown in Table 4.

[0270] [Table 4]

[0271] The sequence of the Sb-aptamer was set with reference to reference 27. In plasmids A and C, the Sb-aptamer was inserted into helix 33a of 16S rRNA in order not to impair the function of 16S rRNA, and in plasmids B and C, the Sb-aptamer was inserted into helix 25 of 23S rRNA in order not to impair the function of 23S rRNA. （参考文献28和29） The base sequence of plasmid A is shown in sequence number 3. Sb-aptamer is encoded at positions 3514 to 3568 of sequence number 3. The base sequence of plasmid B is shown in sequence number 4. Sb-aptamer is encoded at positions 5011 to 5081 of sequence number 4. The base sequence of plasmid C is shown in sequence number 5. Sb-aptamer is encoded at positions 3514 to 3568 and 5066 to 5136 of sequence number 5, respectively.

[0272] Next, SSU having 16S rRNA with Sb-aptamer inserted therein and LSU having 23S rRNA with Sb-aptamer inserted therein were prepared ( Fig. 9 (a) ) to confirm whether they actually have translational activity.

[0273] The production of SSU and confirmation of translation activity were carried out according to the following steps. In the first reaction, 80nM native ribosomes, 0.3nM plasmid A, and 0.05nM of 21 plasmids containing 21 SSU r-protein genes were mixed at respective concentrations in the first reaction solution based on S150 cell extract and reacted at 37°C for 180 minutes. Next, in the second reaction, the translation activity of the newly generated ribosomal SSU was detected by batch analysis using pT7_oSD_or1_LacZ reporter and spectinomycin.

[0274] In addition, the production of LSU and confirmation of translation activity were carried out according to the following steps. In the first reaction, 80nM native ribosomes, 0.9nM plasmid B, and 0.01nM of 31 plasmids containing 33 LSU r-protein genes were mixed at respective concentrations in the first reaction solution based on S150 cell extract and reacted at 37°C for 180 minutes. Then, in the second reaction, the translation activity of the newly generated ribosome LSU was detected by batch analysis using the pT7_wtSD_LacZ reporter in the presence of 1.5mM clindamycin.

[0275] As a result, it was confirmed that both SSU having 16S rRNA with Sb-aptamer inserted therein and LSU having 23S rRNA with Sb-aptamer inserted therein had translation activity ( Fig. 9 (b)).

[0276] 4-2. Production and purification of SSU and LSU having streptavidin-binding aptamers Next, production and purification of SSU having 16S rRNA into which Sb-aptamer was inserted, production and purification of LSU having 23S rRNA into which Sb-aptamer was inserted, and simultaneous production and purification of both were performed.

[0277] The production and purification of SSU having 16S rRNA inserted with Sb-aptamer was carried out according to the following steps. In the first reaction solution based on S150 cell extract, 80nM native ribosomes, 0.3nM plasmid A, and 0.05nM of 21 plasmids containing 21 SSU r-protein genes were mixed at respective concentrations and reacted at 37°C for 180 minutes. The obtained reaction solution 250 μ L, Streptavidin agarose (Thermo Fisher Scientific) 50 μL, and 1.5 mL of ribosome dissociation buffer (20 mM HEPES-KOH, 1 mM magnesium glutamate, 200 mM potassium glutamate, 4 mM DTT, pH = 7.5) were mixed and incubated at 4°C for 16 hours. Next, the streptavidin agarose was washed 7 times with 1 mL of ribosome dissociation buffer. Then, the SSU with the Sb-aptamer was eluted from the streptavidin agarose using a ribosome dissociation buffer containing 25 mM biotin (Nacalai tesque) to purify it.

[0278] The production and purification of LSU having 23S rRNA inserted with Sb-aptamer was carried out according to the following steps. In the first reaction solution based on S150 cell extract, 80nM native ribosomes, 0.9nM plasmid B, and 0.01nM of 31 plasmids containing 33 LSU r-protein genes were mixed at respective concentrations and reacted at 37°C for 180 minutes. Using the obtained reaction solution, LSU having Sb-aptamer was purified under the same conditions as above.

[0279] The simultaneous production and purification of SSU having 16S rRNA inserted with Sb-aptamer and LSU having 23S rRNA inserted with Sb-aptamer were performed according to the following procedure. In the first reaction solution based on S150 cell extract, 80nM native ribosomes, 0.9nM plasmid B, 0.01nM each of 21 plasmids containing 21 SSU r-protein genes, and 0.01nM each of 33 plasmids containing 33 LSU r-protein genes were mixed at respective concentrations and reacted at 37°C for 180 minutes. Using the obtained reaction solution, SSU having Sb-aptamer and LSU having Sb-aptamer were purified under the same conditions as above.

[0280] Using RNeasy Mini Kit, rRNA was extracted from SSUs having 16S rRNA inserted with Sb-aptamer and / or LSUs having 23S rRNA inserted with Sb-aptamer before and after purification, and rRNA was analyzed by electrophoresis. As a result, only a peak of 16S rRNA was observed from the purified SSUs having 16S rRNA inserted with Sb-aptamer, and it was confirmed that SSUs having 16S rRNA inserted with Sb-aptamer were purified with high specificity ( Fig. 9 In addition, in the case of producing LSU having 23S rRNA with Sb-aptamer inserted therein, and in the case of producing SSU having 16S rRNA with Sb-aptamer inserted therein and LSU having 23S rRNA with Sb-aptamer inserted therein simultaneously, it was also confirmed that the LSU was purified with high specificity ( Fig. 9 (c)).

[0281] 4-3. Translation activity of purified ribosomes Next, the newly generated artificial ribosomes containing Sb-aptamers and LSU containing Sb-aptamers were subjected to femtoliter droplet analysis to detect translation activity. μ The results showed that the newly produced artificial ribosomes containing Sb-aptamer SSU and LSU containing Sb-aptamer showed translation activity in the presence of spectinomycin and clindamycin. Fig. 9 (d)), thus confirming that the purified nascent artificial ribosomes are indeed functional.

[0282] 5. References Reference 1: Datsenko, KA et al. One-step inactivation of chromosomalgenes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645 (2000). Reference 2: Boni, IV et al. Ribosome-messenger recognition: mRNA targetsites for ribosomal protein S1. Nucleic Acids Res. 19, 155-162 (1991). Reference 3: Komarova, AV et al. Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation. RNA 8, 1137-1147 (2002). Reference 4: Chen, SS et al. Characterization of the ribosome biogenesislandscape in E.coli using quantitative mass spectrometry. J. Mol. Biol. 425, 767-779 (2013). Reference 5: Sheahan, T. et al. Ribosomal Protein S1 Improves the Protein Yield of an In Vitro Reconstituted Cell-Free Translation System.ACSSynth.Biol.11,1004-1008 (2022). Reference 6: Shilling, PJ et al. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol 3, 214 (2020). Reference 7: Takahashi, S. et al. Translation enhancer improves theribosome liberation from translation initiation. J. Am. Chem. Soc. 135, 13096-13106 (2013). Reference 8: Kwon, Y.-C. et al.High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci.Rep.5,8663 (2015). Reference 9: Jewett, MC et al. In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation. Mol. Syst. Biol. 9, 678 (2013). Reference 10: Fritz, BRet al. The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction. Nucleic AcidsRes.42, 6774-6785 (2014). Reference 11: Kempf, N. et al. A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthes is Systems. Sci.Rep.7, 46753 (2017). Reference 12: Umezawa, H. et al. A NEW ANTIBIOTIC, KASUGSMYCIN.J.Antibiot.18, 101-103 (1965). Reference 13: Schluenzen, F. et al.The antibiotic kasugamycin mimics mRNAnucleotides to destabilize tRNA binding and inhibit canonical translation initiation.Nat.Struct.Mol.Biol.13,871-878 (2006). Reference 14: Berg, S. et al. ilastik: interactive machine learning for (bio)image analysis. Nat. Methods 16, 1226-1232 (2019). Reference 15: Ronneberger, O. et al. U-Net: Convolutional Networks forBiomedical Image Segmentation. in Medical Image Computing and Computer-Assisted Intervention-MICCAI 2015 234-241 (Springer International Publishing, 2015). Reference 16: Howard, J. et al. Fastai: A Layered API for DeepLearning.Information 11,108 (2020). Reference 17: Rackham, O. et al. A network of orthogonal ribosome x mRNApairs. Nat. Chem. Biol. 1, 159-166 (2005). Page 18:Shimojo,M.et al.In vitro reconstitution of functionalsmall ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E.coli.Commun Biol 3,142(2020). Page 19:Sigmund,CDet al.Antibiotic resistance mutations in 16Sand 23S ribosomal RNA genes of Escherichia coli.Nucleic Acids Res.12,4653-4663(1984). Page 20:Rondelez,Y.et al. Microfabricated arrays of femtoliter chambers allow single molecule enzymology. Nat.Biotechnol. Rev. 23,361–365(2005). Publication 21:Schneider,CAet al.NIH Image to ImageJ: 25 years of imageanalysis.Nat.Methods 9,671-675(2012). Page 22:Maitra,A.et al.Bacterial growth laws reflect theevolutionary importance of energy efficiency. Proc.Natl.Acad.Sci.USA 112,406–411(2015). Page 23:Reuveni,S.et al.Optimized ribosomes for autocatalyticproduction.Nature 547,293-297(2017). Page 24:Chumpolkulwong,N.et al.Effects of Escherichia coliribosomal protein S12 mutations on cell-free protein synthesis.Eur.J.Biochem.271,1127-1134(2004). Paper 25:Cochella,L.et al.Isolation of antibiotic resistance mutations in the rRNA by using an in vitro selection system.Proc.Natl.Acad.Sci.USA101,3786-3791(2004). Page 26:Hammerling,MJet al.In vitro ribosome synthesis andevolution through ribosome display.Nat.Commun.11,1108(2020). References 27:Bachler,M.,Schroeder,R.&von Ahsen,U.StreptoTag: a novel method for the isolation of RNA-binding proteins.RNA 5,1509-1516(1999). [ PubMed ] 28:Leonov,AA,Sergiev,PV,Bogdanov,AA,Brimacombe,R.&Dontsova,OAAaffinity purification of ribosomes with a lethal G2655C mutationin 23 S rRNA that affects the translocation.J.Biol.Chem.278.25664–25670(2003). Reference 29: Golovina, A.Y., Bogdanov, A.A., Dontsova, O.A. & Sergiev, P.V. Purification of 30S ribosomal subunit by streptavidin affinity chromatography. Biochimie 92, 914 - 917 (2010).

Claims

1. A method for producing a ribosomal large subunit, characterized in that: A ribosomal large subunit is produced in a cell-free protein synthesis system, the production method comprising the following steps: In the cell-free protein synthesis system, ribosomal large subunit protein and ribosomal large subunit RNA are assembled to form the ribosomal large subunit. In the cell-free protein synthesis system, at least one of the ribosomal large subunit proteins is produced from a template nucleic acid encoding a ribosomal large subunit protein.

2. The manufacturing method according to claim 1, wherein: At least one of the ribosomal large subunit RNAs is produced from a template DNA encoding the ribosomal large subunit RNA in the cell-free protein synthesis system.

3. The manufacturing method according to claim 1 or 2, wherein: The large ribosomal subunit is derived from Escherichia coli.

4. The manufacturing method according to claim 3, wherein: The ribosomal large subunit protein constituting the ribosomal large subunit does not include the 50S r-protein L11 but includes 32 or less 50S r-proteins.

5. The manufacturing method according to claim 1 or 2, wherein: The large ribosomal subunit RNA is subjected to a mutation that confers drug resistance.

6. The manufacturing method according to claim 1 or 2, wherein: In the cell-free protein synthesis system, all ribosomal large subunit proteins to be assembled are produced from template nucleic acids encoding ribosomal large subunit proteins.

7. A method for producing a polymer in a cell-free synthesis system, characterized in that Including the following processes: A first step, wherein a ribosomal large subunit protein and a ribosomal large subunit RNA are assembled in a cell-free protein synthesis system to produce a ribosomal large subunit; and A second step, wherein a polymer is synthesized from a template nucleic acid encoding a sequence of the polymer in a cell-free synthesis system containing a ribosome having a ribosomal large subunit obtained in the first step; In the cell-free protein synthesis system of the first step, at least one of the ribosomal large subunit proteins is produced from a template nucleic acid encoding a ribosomal large subunit protein in the cell-free protein synthesis system.

8. A method for producing a ribosomal small subunit, characterized in that: A ribosomal small subunit is produced in a cell-free protein synthesis system, the production method comprising the following steps: In the cell-free protein synthesis system, ribosomal small subunit protein and ribosomal small subunit RNA are assembled to form the ribosomal small subunit, In the cell-free protein synthesis system, at least two of the small ribosomal subunit proteins are produced from a template nucleic acid encoding a small ribosomal subunit protein.

9. The method for producing a ribosomal small subunit according to claim 8, wherein: The ribosomal small subunit RNA is produced from a template DNA encoding the ribosomal small subunit RNA in the cell-free protein synthesis system.

10. The manufacturing method according to claim 8 or 9, wherein: The small ribosomal subunit is derived from Escherichia coli.

11. The manufacturing method according to claim 8 or 9, wherein: The anti-SD sequence of the small ribosomal subunit RNA is set to be able to recognize an SD sequence that is not recognized by the anti-SD sequence of the small ribosomal subunit RNA of the ribosome used for translation in the cell-free protein synthesis system.

12. The manufacturing method according to claim 8 or 9, wherein: A drug resistance-conferring mutation is performed on at least one of the small ribosomal subunit RNA and the small ribosomal subunit protein.

13. The manufacturing method according to claim 8 or 9, wherein: In the cell-free protein synthesis system, all the small ribosomal subunit proteins to be assembled are produced from template nucleic acids encoding the small ribosomal subunit proteins.

14. A method for producing a polymer in a cell-free synthesis system, characterized in that: Including the following processes: A first step, wherein a ribosomal small subunit protein and a ribosomal small subunit RNA are assembled in a cell-free protein synthesis system to produce a ribosomal small subunit; and The second step comprises synthesizing a polymer from a template nucleic acid encoding a sequence of the polymer in a cell-free synthesis system containing a ribosome having a ribosomal small subunit obtained in the first step, In the cell-free protein synthesis system of the first step, at least two of the small ribosomal subunit proteins are produced from a template nucleic acid encoding a small ribosomal subunit protein.

15. A method for producing ribosomes, characterized in that: A ribosome is produced in a cell-free protein synthesis system, the production method comprising the following steps: In the cell-free protein synthesis system, the assembly of the large ribosomal subunit protein and the large ribosomal subunit RNA, as well as the assembly of the small ribosomal subunit protein and the small ribosomal subunit RNA, is carried out to form ribosomes. In the cell-free protein synthesis system, at least one of the large ribosomal subunit protein and the small ribosomal subunit protein is produced from a template nucleic acid encoding the protein.

16. The manufacturing method according to claim 15, wherein: The ribosomes are derived from Escherichia coli.

17. The manufacturing method according to claim 15 or 16, wherein: In the cell-free protein synthesis system, all ribosomal large subunit proteins to be assembled are produced from template nucleic acids encoding ribosomal large subunit proteins.

18. The manufacturing method according to claim 15 or 16, wherein: In the cell-free protein synthesis system, all the small ribosomal subunit proteins to be assembled are produced from template nucleic acids encoding the small ribosomal subunit proteins.

19. The manufacturing method according to claim 15 or 16, wherein: In the cell-free protein synthesis system, at least one of the ribosomal large subunit RNA and the ribosomal small subunit RNA is produced from a template DNA encoding the RNA.

20. A method for producing a polymer in a cell-free synthesis system, characterized in that: Including the following processes: The first step comprises assembling a ribosomal large subunit protein and a ribosomal large subunit RNA, and assembling a ribosomal small subunit protein and a ribosomal small subunit RNA in a cell-free protein synthesis system to form a ribosome; and The second step comprises synthesizing a polymer from a template nucleic acid encoding a sequence of the polymer in a cell-free synthesis system containing the ribosomes obtained in the first step, In the cell-free protein synthesis system of the first step, at least one of the large ribosomal subunit protein and the small ribosomal subunit protein is produced from a template nucleic acid encoding the protein.

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