Cell-free protein synthesis system, reaction mixture for cell-free protein synthesis system, and method for preparing proteins

Abstract

Disclosed herein are cell-free protein synthesis systems, reaction mixtures for cell-free protein synthesis systems, and methods for preparing proteins of interest.

Description

[Background technology]

[0001] Cell-free protein synthesis (CFPS) offers many advantages over cell-based protein synthesis, including reduced processing time and increased overall yield of functional, soluble, full-length proteins. Additionally, CFPS is less sensitive to toxic proteins than cell-based protein synthesis and is adaptable to high-throughput experiments.

[0002] Commercially available CFPS mixtures typically require complex and expensive feedstocks, such as free amino acids, free ribonucleotides, and free NADH / NADPH. While commercial CFPSs typically emphasize reaction rate, their yields are often limited by the rate-limiting feedstock. This is because the feedstock must be generated and regenerated before it can be converted into energy and / or protein products. In addition, commercial CFPSs often rely on systems that must be stored at lower temperatures, such as -20°C. Summary of the Invention [Problem to be solved by the invention]

[0003] In contrast, the present application discloses a novel CFPS system, including its novel reaction mixture, that uses simpler and less expensive feedstocks and avoids many of the problems faced by commercially available CFPSs (e.g., wasteful consumption of amino acids in side reactions, regeneration of NADH / NADPH), providing cost-saving benefits. In addition, the novel CFPS system and reaction mixture can be stored at 4°C and / or room temperature, thereby avoiding the inconvenience and costs associated with storage at low temperatures. [Means for solving the problem]

[0004] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0005] [Figure 1] The effect of extract and mixture conditions, as well as temperature, on CFPS deGFP protein yield is shown. "Min" and "Com" refer to "minimal" and "commercially available," respectively, and "CE" refers to cell extract. [Figure 2] 1 shows the effect of removing individual reaction mixture components on CFPS deGFP protein yield when minimal cell extract is used. [Figure 3] 1 shows the effect of sodium chloride concentration on CFPS protein yield when utilizing minimal cell extracts from V. natriegens. DETAILED DESCRIPTION OF THE INVENTION

[0006] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any claimed subject matter. To the extent that any material incorporated herein by reference is inconsistent with the language of the present disclosure, the language controls. In this application, the use of the singular includes the plural unless expressly stated otherwise. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0007] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is present in at least some embodiments, but not necessarily all embodiments of the invention.

[0008] As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" also includes the exact amount. Thus, "about 5 μL" means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error. For example, the term

[0009] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way.

[0010] As used herein, a "cell-free" or "CF" system or component thereof is one that performs its intended function (e.g., protein synthesis) primarily by utilizing non-viable cell aspects of the system or component (e.g., supernatant recovered from lysed bacteria). A "cell-free" system or component thereof may contain residual amounts of viable cells.

[0011] As used herein, a "minimal medium" is a medium containing only the minimum set of nutrients required for bacterial growth. The nutrients may vary depending on the bacterial species and are well known to those skilled in the art for a given species. In one embodiment, a "minimal medium" consists essentially of (a) at least one carbon source (e.g., a sugar such as glucose, a polyol such as glycerol, or a dicarboxylic acid such as succinic acid), (b) at least one of an ammonium source (e.g., ammonium sulfate), a phosphate source (e.g., monopotassium phosphate or dipotassium phosphate), a sulfur source, a magnesium source (e.g., magnesium sulfate), and a sodium salt (e.g., sodium succinate), and (c) water. A "minimal medium" may contain elements for structural support of bacterial culture but may be free of elements that do not function as nutrients for bacteria (e.g., agar). Preferably, a "minimal medium" does not contain vitamins or coenzymes. Preferably, a "minimal medium" does not contain amino acids or proteins.

[0012] As used herein, "DNA" or "RNA" encoding a protein of interest refers to one or more deoxyribonucleic acids or ribonucleic acids having codons or anticodons that encode at least a portion of the protein of interest. The protein of interest may be encoded by two or more deoxyribonucleic acids or ribonucleic acids, or a combination thereof. The DNA or RNA may be circular or linear, and the DNA may be single-stranded or double-stranded. In some embodiments, the DNA or RNA may be a plasmid.

[0013] As used herein, a "free amino acid" refers to an amino acid that is not part of a polypeptide or protein.

[0014] As used herein, "free nucleotide" refers to a nucleotide that is not part of a polynucleotide and that includes the corresponding nucleoside. Exemplary polynucleotides include, but are not limited to, plasmids, genomic DNA, and genomic RNA, which are not encompassed by "free nucleotides." A "nucleotide" refers to a compound that includes a nucleoside moiety and a phosphate moiety. Exemplary nucleotides include adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), and guanosine monophosphate (GMP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (DTP), and ATP-dependent nucleotides. Deoxycytidine diphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine diphosphate (dADP), thymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP), and deoxyguanosine monophosphate (dGMP).

[0015] As used herein, "free NADH / NADPH" refers to any combination and ratio of free oxidized and reduced nicotinamide adenine dinucleotide and free oxidized and reduced nicotinamide adenine dinucleotide phosphate.

[0016] As used herein, "maintaining" a cell-free protein synthesis system at a certain temperature refers to maintaining the cell-free protein synthesis system in a space (e.g., a room) at a certain temperature for a sufficient amount of time to carry out cell-free protein synthesis. One or more portions of the cell-free protein synthesis system may have a temperature that is higher or lower than the certain temperature of the space.

[0017] While various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein for clarity in the context of separate embodiments, the invention may also be practiced in a single embodiment.

[0018] Cell-free protein synthesis system We have developed a cell-free protein synthesis system that involves cell extracts from bacteria grown in minimal medium and does not require the addition of amino acids or nucleotides, and which appears to function through the use of the aerobic respiratory cycle to produce these components.

[0019] Thus, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria.

[0020] In some embodiments, the pH of the cell-free protein synthesis system is about 7.0 to about 8.0. In some embodiments, the pH of the cell-free protein synthesis system is 7.1 to 7.9. In some embodiments, the pH of the cell-free protein synthesis system is 7.2 to 7.8. In some embodiments, the pH of the cell-free protein synthesis system is 7.3 to 7.7. In some embodiments, the pH of the cell-free protein synthesis system is 7.4 to 7.6. In some embodiments, the pH of the cell-free protein synthesis system is about 7.5. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when present in the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5.

[0021] In some embodiments, the cell-free protein synthesis system is maintained at a temperature below about 30°C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature of about 20°C to about 30°C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature of about 21°C to about 29°C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature of about 22°C to about 28°C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature of about 23°C to about 27°C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature of about 24°C to about 36°C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature greater than -20°C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature of about 25°C. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and wherein the cell-free protein synthesis system is maintained at a temperature of less than about 30°C.

[0022] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when present in the lysed bacteria; the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5; and the cell-free protein synthesis system is maintained at a temperature of less than about 30°C.

[0023] In some embodiments, the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0024] In some embodiments, the reaction mixture further comprises at least two of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0025] In some embodiments, the reaction mixture further comprises at least three of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0026] In some embodiments, the reaction mixture further comprises at least four of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0027] In some embodiments, the reaction mixture further comprises at least five of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0028] In some embodiments, the reaction mixture further comprises at least six of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0029] In some embodiments, the reaction mixture further comprises at least seven of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0030] In some embodiments, the reaction mixture further comprises (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and optionally (h) a buffer.

[0031] In some embodiments, the reaction mixture further comprises (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0032] In some embodiments, the reaction mixture comprises NaCl. NaCl may be present at 50-250 mM, e.g., 100-200 mM, e.g., 100 mM. Such NaCl concentrations are advantageous when cell extracts derived from V. natriegens are to be used.

[0033] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5; the cell-free protein synthesis system is maintained at a temperature of less than about 30°C; and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0034] In some embodiments, the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. In some embodiments, the molecular crowder is PEG 400. In some embodiments, the molecular crowder is PEG 1500. In some embodiments, the molecular crowder is PEG 3350. In some embodiments, the molecular crowder is PEG 4000. In some embodiments, the molecular crowder is PEG 6000. In some embodiments, the molecular crowder is PEG 8000. In some embodiments, the molecular crowder is maltodextrin. In some embodiments, the molecular crowder is Ficoll 70. In some embodiments, the molecular crowder is Ficoll 400. In some embodiments, the molecular crowder is dextran. In some embodiments, the molecular crowder is serum albumin. In some embodiments, the molecular crowder is bovine serum albumin. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin.

[0035] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, or about 7. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder, when present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin.

[0036] In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is about 0 to 40 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is about 2.5 to 37.5 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is about 5 to 35 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is about 7.5 to 32.5 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is about 10 to 30 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is about 12.5 to 27.5 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is about 15 to 25 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is about 17.5 to 22.5 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is 20 g / L. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when present in the lysed bacteria, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the concentration of the molecular crowder when present in the cell-free protein synthesis system is about 0 to 40 g / L.

[0037] In some embodiments, provided herein is a cell-free protein synthesis system, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder, if present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and wherein the concentration of the molecular crowder in the cell-free protein synthesis system, if present, is about 0-40 g / L.

[0038] In some embodiments, the carbon source comprises a sugar. In some embodiments, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. In some embodiments, the carbon source comprises glucose. In some embodiments, the carbon source comprises fructose. In some embodiments, the carbon source comprises galactose. In some embodiments, the carbon source comprises sucrose. In some embodiments, the carbon source comprises lactose. In some embodiments, the carbon source comprises gluconate. In some embodiments, the carbon source comprises starch. In some embodiments, the carbon source comprises maltodextrin. In some embodiments, the carbon source comprises maltose. Alternatively, in some embodiments, the carbon source does not comprise maltose. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose.

[0039] In some embodiments, a cell-free protein synthesis system is provided herein, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or is about 7.5, the cell-free protein synthesis system comprises a reaction mixture maintained at a temperature of less than about 30°C, the molecular crowder, when present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran and serum albumin, optionally bovine serum albumin, the concentration of the molecular crowder in the cell-free protein synthesis system is about 0-40 g / L, and the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin and maltose.

[0040] In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 2 to 47 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 4 to 44 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 6 to 41 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 8 to 38 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 10 to 35 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 12 to 32 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 14 to 29 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 16 to 36 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is about 18-33 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is 20 g / L. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the concentration of the carbon source in the cell-free protein synthesis system, if present, is about 0-50 g / L.

[0041] In some embodiments, provided herein is a cell-free protein synthesis system, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the cell-free protein synthesis system is maintained at a temperature of less than about 30° C., and the reaction mixture comprises: (a) a molecular crowder; (b) a carbon source; (c) a molecular crowder; (f) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder, if present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin, and the concentration of the molecular crowder, if present, is about 0-40 g / L, and the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, and the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L.

[0042] In some embodiments, the ammonium source comprises ammonium sulfate. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 2 to 20 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 3 to 19 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 4 to 18 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 5 to 17 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 6 to 16 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 7 to 16 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 8 to 16 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 9 to 15 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 10 to 14 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is about 11-13 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is 12 mM. In some embodiments, a cell-free protein synthesis system is provided herein, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the ammonium source comprises ammonium sulfate.In some embodiments, provided herein is a cell-free protein synthesis system, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the concentration of the ammonium source, if present in the cell-free protein synthesis system, is about 2-20 mM.

[0043] In some embodiments, provided herein is a cell-free protein synthesis system, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the cell-free protein synthesis system is maintained at a temperature of less than about 30° C., and the reaction mixture comprises: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; and (f) a phosphorus source. and (h) at least one of (a) an acid source; and (b) a sulfur source; and (c) a buffer solution, wherein the molecular crowder, if present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin, and the concentration of the molecular crowder, if present in the cell-free protein synthesis system, is about 0-40 g / L; the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, and the concentration of the carbon source, if present in the cell-free protein synthesis system, is about 0-50 g / L; and the ammonium source, if present, comprises ammonium sulfate, and the concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM.

[0044] In some embodiments, the potassium source is selected from potassium glutamate and potassium gluconate. In some embodiments, the potassium source comprises potassium glutamate and potassium gluconate. In some embodiments, the potassium source comprises potassium glutamate. In some embodiments, the potassium source comprises potassium gluconate. In some embodiments, the potassium source comprises potassium gluconate. In some embodiments, a cell-free protein synthesis system is provided herein, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the potassium source, if present, is selected from potassium glutamate and potassium gluconate.

[0045] In some embodiments, provided herein is a cell-free protein synthesis system, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the cell-free protein synthesis system is maintained at a temperature of less than about 30° C., and the reaction mixture comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer. the molecular crowder, if present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; the concentration of the molecular crowder, if present, is about 0-40 g / L; the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source, if present, is about 0-50 g / L; the ammonium source, if present, comprises ammonium sulfate; the concentration of the ammonium source, if present, is 2-20 mM; and the potassium source, if present, is selected from potassium glutamate and potassium gluconate.

[0046] In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 11 to 240 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 12 to 210 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 13 to 180 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 14 to 150 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 15 to 120 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 16 to 90 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 17 to 60 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 18 to 30 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 18 to 28 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 18 to 26 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 18 to 24 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 19 to 24 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 19 to 22 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 19 to 21 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 20 mM.(e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the potassium source, if present, is selected from potassium glutamate and potassium gluconate, and wherein the concentration of potassium glutamate, if present in the cell-free protein synthesis system, is about 10 to 270 mM.

[0047] In some embodiments, provided herein is a cell-free protein synthesis system, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the cell-free protein synthesis system is maintained at a temperature of less than about 30° C., and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder When present, the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; when present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L; when present in the cell-free protein synthesis system, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present in the cell-free protein synthesis system, the concentration of the carbon source is about 0-50 g / L; when present in the cell-free protein synthesis system, the ammonium source comprises ammonium sulfate; when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2-20 mM; and when present in the cell-free protein synthesis system, the potassium source is selected from potassium glutamate and potassium gluconate; when present in the cell-free protein synthesis system, the concentration of the potassium glutamate is about 10-270 mM.

[0048] In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 15 to 240 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 20 to 230 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 25 to 220 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 30 to 210 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 35 to 200 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 40 to 190 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 45 to 180 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 50 to 170 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 55 to 160 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 60 to 150 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 70 to 140 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 80 to 130 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 90 to 120 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 95 to 110 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 95 to 105 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 100 mM.(f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the potassium source, if present, is selected from potassium glutamate and potassium gluconate, and wherein the concentration of potassium gluconate, if present in the cell-free protein synthesis system, is about 10 to 250 mM.

[0049] In some embodiments, provided herein is a cell-free protein synthesis system, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and wherein the pH of the cell-free protein synthesis system is about 7. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, and the molecular crowder, when present, is selected from the group consisting of PEG 400 to 8000, maltodextrin, PEG 400 to 80 ... the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the ammonium source, if present, comprises ammonium sulfate; the ammonium source, if present, is selected from potassium glutamate and potassium gluconate; the potassium glutamate, if present, is selected from potassium glutamate and potassium gluconate; the potassium gluconate, if present, is selected from potassium glutamate and potassium gluconate; the potassium gluconate, if present, is selected from potassium glutamate and potassium gluconate;

[0050] In some embodiments, the magnesium source comprises magnesium glutamate. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 0.5 to 9.25 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 1 to 8.5 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 1.5 to 7.75 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 2 to 7 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 2.5 to 6.25 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 3 to 5.5 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 3.5 to 4.75 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 3.5 to 4.5 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 3.75 to 4.25 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 4 mM. In some embodiments, a cell-free protein synthesis system is provided herein, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the magnesium source, when present, comprises magnesium glutamate.(e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the magnesium source, when present, comprises magnesium glutamate, and wherein the concentration of the magnesium source, when present in the cell-free protein synthesis system, is from 0 to about 10 mM.

[0051] In some embodiments, a cell-free protein synthesis system is provided herein, the cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2, or about 8.0. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder, when present, is selected from the group consisting of PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, and optionally, PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin. When present in the cell-free protein synthesis system, the molecular crowder has a concentration of about 0 to 40 g / L; when present, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present, the carbon source has a concentration of about 0 to 50 g / L; and when present, the ammonium source comprises ammonium sulfate; when present, the ammonium source comprises ammonium sulfate. When present, the concentration of the potassium glutamate is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; when present in the cell-free protein synthesis system, the concentration of the potassium glutamate is about 10 to 270 mM; when present in the cell-free protein synthesis system, the concentration of the potassium gluconate is about 10 to 250 mM; when present, the magnesium source comprises magnesium glutamate; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM.

[0052] In some embodiments, the phosphate source comprises potassium phosphate. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 6 to 22 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 7 to 20.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 8 to 19 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 9 to 17.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 10 to 16 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 11 to 14.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 12 to 14 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 12.5 to 13.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 13 mM. In some embodiments, the cell-free protein synthesis system includes: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further includes at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the phosphate source includes potassium phosphate.In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the concentration of the phosphate source in the cell-free protein synthesis system, if present, is about 5 to about 23.5 mM.

[0053] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, or about 7.3 to 7.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin, and the cell-free protein synthesis system is maintained at a temperature of less than about 30°C. When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L; the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present in the cell-free protein synthesis system, the concentration of the carbon source is about 0-50 g / L; the ammonium source, if present, comprises ammonium sulfate; when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2-20 mM; and when present in the cell-free protein synthesis system, the potassium source, if present, is potassium glutamate and gluconate. When present in the cell-free protein synthesis system, the potassium glutamate concentration is about 10 to 270 mM; when present in the cell-free protein synthesis system, the potassium gluconate concentration is about 10 to 250 mM; when present in the cell-free protein synthesis system, the magnesium source comprises magnesium glutamate; when present in the cell-free protein synthesis system, the magnesium source concentration is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the phosphate source comprises potassium phosphate; when present in the cell-free protein synthesis system, the phosphate source concentration is about 5 to about 23.5 mM.

[0054] In some embodiments, the sulfur source comprises ammonium sulfate. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 3 to 19 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 4 to 18 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 5 to 17 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 6 to 16 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 7 to 15 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 8 to 15 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 9 to 14 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 10 to 14 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 11 to 13 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 11.5 to 12.5 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 12 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the sulfur source comprises ammonium sulfate.In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the concentration of the sulfur source, if present in the cell-free protein synthesis system, is about 2-20 mM.

[0055] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, or about 7.3 to 7.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder, when present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and the cell-free protein synthesis system is maintained at a temperature of less than about 30°C. When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L, and the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. When present in the cell-free protein synthesis system, the concentration of the carbon source is about 0-50 g / L, and the ammonium source, when present, comprises ammonium sulfate. When present in the cell-free protein synthesis system, the concentration of the ammonium source is 2-20 mM, and the potassium source, when present, is potassium glutamate. and potassium gluconate, wherein when present in the cell-free protein synthesis system, the concentration of potassium glutamate is about 10 to 270 mM; when present in the cell-free protein synthesis system, the concentration of potassium gluconate is about 10 to 250 mM; when present in the cell-free protein synthesis system, the magnesium source comprises magnesium glutamate; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the phosphate source comprises potassium phosphate; when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The sulfur source, if present, includes ammonium sulfate, and the concentration of the sulfur source, if present in the cell-free protein synthesis system, is 2-20 mM.

[0056] In some embodiments, the cell-free protein synthesis system comprises a buffer. In some embodiments, the buffer comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 50 to 300 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 55 to 280 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 60 to 260 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 65 to 240 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 70 to 220 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 75 to 200 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 80 to 180 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 85 to 160 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 90 to 140 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 95 to 120 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 95 to 110 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 95 to 105 mM. In some embodiments, the buffer concentration in the cell-free protein synthesis system is 100 mM.In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the buffer, if present, comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the buffer solution, if present in the cell-free protein synthesis system, has a concentration of 50 to 300 mM.

[0057] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, or about 7.3 to 7.7. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder, when present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and the cell-free protein synthesis system is maintained at a temperature of less than about 30°C. When present, the concentration of the molecular crowder is about 0-40 g / L; when present, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present, the concentration of the carbon source is about 0-50 g / L; when present, the ammonium source comprises ammonium sulfate; when present, the concentration of the ammonium source is 2-20 mM; and when present, the potassium source is potassium glutamate. The magnesium source, if present, is selected from sodium and potassium gluconate, and when present in the cell-free protein synthesis system, the potassium glutamate concentration is about 10 to 270 mM. When present in the cell-free protein synthesis system, the potassium gluconate concentration is about 10 to 250 mM. When present in the cell-free protein synthesis system, the magnesium source comprises magnesium glutamate, and when present in the cell-free protein synthesis system, the magnesium source concentration is 0 to about 10 mM. When present in the cell-free protein synthesis system, the phosphate source comprises potassium phosphate, and when present in the cell-free protein synthesis system, the phosphate source concentration is about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, and if present in the cell-free protein synthesis system, the sulfur source has a concentration of 2 to 20 mM. The buffer, if present, comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, and if present in the cell-free protein synthesis system, the buffer has a concentration of 50 to 300 mM.

[0058] In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 12% to 66% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 14% to 62% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 16% to 58% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 18% to 54% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 20% to 50% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 22% to 46% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 24% to 42% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 26% to 38% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 28% to 34% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 29% to 32% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 29% to 31% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 30% (v / v). In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when present in the lysed bacteria, and the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v).

[0059] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, or about 7.3. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and wherein the cell-free protein synthesis system is maintained at a temperature of less than about 30°C; and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and When present, the concentration of the molecular crowder is about 0-40 g / L; when present, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present in the cell-free protein synthesis system, the concentration of the carbon source is about 0-50 g / L; when present, the ammonium source comprises ammonium sulfate; when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2-20 mM; when present, the potassium source is potassium glutamate and and potassium gluconate, wherein when present in the cell-free protein synthesis system, the concentration of potassium glutamate is about 10 to 270 mM; when present in the cell-free protein synthesis system, the concentration of potassium gluconate is about 10 to 250 mM; when present in the cell-free protein synthesis system, the magnesium source comprises magnesium glutamate, wherein when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the phosphate source comprises potassium phosphate, wherein when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate. If present in the cell-free protein synthesis system, the sulfur source concentration is 2-20 mM. If present in the cell-free protein synthesis system, the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. If present in the cell-free protein synthesis system, the buffer concentration is 50-300 mM. If present in the cell-free protein synthesis system, the cell-free cell extract concentration is 10%-70% (v / v).

[0060] In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 2 to 90 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 3 to 80 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 4 to 70 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 5 to 60 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 6 to 50 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 7 to 40 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 8 to 30 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 9 to 20 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 9 to 15 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 9.5 to 12.5 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 9.5 to 11.5 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 9.5 to 11 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 10 μg / mL.In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL.

[0061] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and wherein the cell-free protein synthesis system is maintained at a temperature of less than about 30°C; and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; When present in the protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L; when present, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L; when present, the ammonium source comprises ammonium sulfate; when present, the concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM; and when present, the potassium source is potassium glutamate. and potassium gluconate, and when present in the cell-free protein synthesis system, the concentration of potassium glutamate is about 10 to 270 mM, and the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; the magnesium source, when present, comprises magnesium glutamate, and when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and the phosphate source, when present, comprises potassium phosphate, and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 5 mM, the sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM, the buffer, if present, comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), and the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL.

[0062] In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.9 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.8 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.7 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.6 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.5 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.4 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.3 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.2 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.1 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 1 mM.In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.9 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.8 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.7 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.6 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.5 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.4 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.3 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.2 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.1 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.05 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.025 mM.In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.01 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, wherein the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM.

[0063] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.9 to 8.9, or about 8.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and wherein the cell-free protein synthesis system is maintained at a temperature of less than about 30°C; and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and When present in the synthesis system, the concentration of the molecular crowder is about 0-40 g / L; when present, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present in the cell-free protein synthesis system, the concentration of the carbon source is about 0-50 g / L; when present, the ammonium source comprises ammonium sulfate; when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2-20 mM; and when present, the potassium source is glutamine. The magnesium source, if present, is selected from potassium glutamate and potassium gluconate, and the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM. When present in the cell-free protein synthesis system, the concentration of potassium gluconate is about 10 to 250 mM. When present in the cell-free protein synthesis system, the magnesium source comprises magnesium glutamate. When present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM. When present in the cell-free protein synthesis system, the phosphate source comprises potassium phosphate. When present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 5 mM, the sulfur source, if present, includes ammonium sulfate. If present, the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM. If present, the buffer includes a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. The concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM. The concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v). The concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL. The cell-free protein synthesis system contains free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM.

[0064] In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.5 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.4 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.3 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.2 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.1 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 1 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.9 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.8 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.7 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.6 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.5 mM.In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.25 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.125 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.1 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.075 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to lysed bacteria, and the concentration of free nucleotides in the cell-free protein synthesis system is less than 0.05 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.025 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, wherein the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM.

[0065] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and wherein the cell-free protein synthesis system is maintained at a temperature of less than about 30°C; and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; When present in the protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L, and the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. When present in the cell-free protein synthesis system, the concentration of the carbon source is about 0-50 g / L, and the ammonium source, when present, comprises ammonium sulfate. The concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM, and the potassium source, when present, is glutamic acid. The cell-free protein synthesis system is selected from potassium and potassium gluconate, and the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM. When present in the cell-free protein synthesis system, the concentration of potassium gluconate is about 10 to 250 mM. When present in the cell-free protein synthesis system, the magnesium source comprises magnesium glutamate. When present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM. When present in the phosphate source, the phosphate source comprises potassium phosphate. The concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23 mM.The cell-free protein synthesis system contains 5 mM sulfur, if present, and ammonium sulfate, and the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM. The buffer, if present, contains 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, and the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM. The concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v). The concentration of DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL. The cell-free protein synthesis system contains free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM.

[0066] In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.35 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.3 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.25 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.2 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.15 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.1 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.08 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.06 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.04 mM.In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.02 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.01 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, wherein the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, and wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM.

[0067] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder, when present, is selected from the group consisting of PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. the concentration of the molecular crowder in the cell-free protein synthesis system is about 0 to 40 g / L; the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L; the ammonium source, when present, comprises ammonium sulfate; the concentration of the ammonium source in the cell-free protein synthesis system is 2 to 20 mM; and the potassium source, when present, When present, the source is selected from potassium glutamate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM, the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM, and the phosphate source comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, and the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM. The buffer, if present, comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, and the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM. The concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v). The concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system is 1 to 100 μM. The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM.

[0068] In some embodiments, the cell-free protein synthesis system does not contain tryptone. In some embodiments, the cell-free protein synthesis system does not contain yeast extract. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when present in the lysed bacteria, and the cell-free protein synthesis system does not contain tryptone. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when present in the lysed bacteria, and the cell-free protein synthesis system does not contain yeast extract.

[0069] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and wherein the cell-free protein synthesis system is maintained at a temperature of less than about 30°C. The concentration of the molecular crowder present in the protein synthesis system is about 0-40 g / L, the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L, the ammonium source, if present, comprises ammonium sulfate, the concentration of the ammonium source, if present in the cell-free protein synthesis system, is 2-20 mM, and the potassium source, if present, is glutamic acid. The phosphate source is selected from potassium phosphate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM, and when present, the concentration of potassium gluconate is about 10 to 250 mM. The magnesium source comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM. The phosphate source, when present, comprises potassium phosphate, and the concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, and the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM. The buffer, if present, comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, and the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM. The concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v). The concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL. The cell-free protein synthesis system comprises a lysate of 50 ... The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, the concentration of the free amino acids in the cell-free protein synthesis system being less than 2 mM, the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, the concentration of nucleotides in the cell-free protein synthesis system being less than 1.5 mM, the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, the concentration of free NADH / NADPH in the cell-free protein synthesis system being less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, and the cell-free protein synthesis system does not contain yeast extract.

[0070] Minimal medium In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria. In some embodiments, the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least two of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least three of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least four of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least five of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least six of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least seven of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate.In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate.

[0071] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L, the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L, the ammonium source, when present, comprises ammonium sulfate, the concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM, and the potassium ... ammonium sulfate. The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of the potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of the potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM, the buffer, if present, comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL, the cell-free protein synthesis system containing free amino acids endogenous to lysed bacteria, the concentration of the free amino acids in the cell-free protein synthesis system being 50 to 300 mM, is less than 2 mM, the cell-free protein synthesis system contains free nucleotides endogenous to lysed bacteria, the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM, the cell-free protein synthesis system contains free NADH / NADPH endogenous to lysed bacteria, the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate.

[0072] In some embodiments, the carbon source is selected from D-glucose and glycerol. In some embodiments, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v). In some embodiments, the concentration of D-glucose in the minimal medium is 0.2% to 2% (w / v). In some embodiments, the concentration of D-glucose in the minimal medium is 0.3% to 1.5% (w / v). In some embodiments, the concentration of D-glucose in the minimal medium is 0.4% to 1% (w / v). In some embodiments, the concentration of D-glucose in the minimal medium is 0.4% to 0.8% (w / v). In some embodiments, the concentration of D-glucose in the minimal medium is 0.4% to 0.6% (w / v). In some embodiments, the concentration of D-glucose in the minimal medium is 0.5% (w / v). In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, and the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v).

[0073] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder, when present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L; the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L; the ammonium source, when present, comprises ammonium sulfate; when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2-20 mM; and when present, the potassium source is the magnesium source, when present, comprises magnesium glutamate; the concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM; the phosphate source, when present, comprises potassium phosphate; the concentration of the phosphate source in the cell-free protein synthesis system is 5 to about 23 mM.The concentration of the cell-free protein synthesis system is 5 mM, the sulfur source includes ammonium sulfate, and the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM. The buffer, if present, includes a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, and the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM. The concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v). The concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL. The cell-free protein synthesis system includes free amino acids endogenous to lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides present in lysed bacteria, the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM, the cell-free protein synthesis system contains free NADH / NADPH present in lysed bacteria, the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, the carbon source is selected from D-glucose and glycerol, and the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v).

[0074] In some embodiments, the carbon source is selected from D-glucose and glycerol. In some embodiments, the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 0.5% to 22.5% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 0.75% to 20% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 1% to 17.5% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 1.25% to 15% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 1.5% to 12.5% ​​(w / v). In some embodiments, the concentration of glycerol in the minimal medium is 1.75% to 10% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 2% to 7.5% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 2.25% to 5% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 2.25% to 3.75% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 2.25% to 2.75% (w / v). In some embodiments, the concentration of glycerol in the minimal medium is 2.5% (w / v). In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, and the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v).

[0075] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder, when present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0 to 40 g / L; the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L; the ammonium source, when present, comprises ammonium sulfate; when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM; and the potassium source is selected from glutathione. When present in the cell-free protein synthesis system, the potassium glutamate concentration is about 10 to 270 mM, and the potassium gluconate concentration in the cell-free protein synthesis system is about 10 to 250 mM. When present, the magnesium source comprises magnesium glutamate, and the magnesium source concentration in the cell-free protein synthesis system is 0 to about 10 mM. When present, the phosphate source comprises potassium phosphate, and the phosphate source concentration in the cell-free protein synthesis system is about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM, the buffer, if present, comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL, the cell-free protein synthesis system containing free amino acids endogenous to the lysed bacteria, the concentration of the free amino acids in the cell-free protein synthesis system being less than 2 mM, the cell-free protein synthesis system containing free nucleotides endogenous to the lysed bacteria, The concentration of nucleotides in the cellular protein synthesis system is less than 1.5 mM, the cell-free protein synthesis system contains free NADH / NADPH endogenous to lysed bacteria, the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, the carbon source being selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium being 0.1% to 2.5% (w / v), and the concentration of glycerol in the minimal medium being 0.25% to 25% (w / v).

[0076] In some embodiments, the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v). In some embodiments, the concentration of sodium succinate in the minimal medium is 0.1% to 4% (w / v). In some embodiments, the concentration of sodium succinate in the minimal medium is 0.15% to 3% (w / v). In some embodiments, the concentration of sodium succinate in the minimal medium is 0.2% to 2% (w / v). In some embodiments, the concentration of sodium succinate in the minimal medium is 0.25% to 1% (w / v). In some embodiments, the concentration of sodium succinate in the minimal medium is 0.25% to 0.75% (w / v). In some embodiments, the concentration of sodium succinate in the minimal medium is 0.25% to 0.5% (w / v). In some embodiments, the concentration of sodium succinate in the minimal medium is 0.375% (w / v). Provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v).

[0077] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and wherein the cell-free protein synthesis system is maintained at a temperature of less than about 30°C; and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; When present in the protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L; when present, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L; when present, the ammonium source comprises ammonium sulfate; when present, the concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM; and when present, the potassium source is potassium glutamate. The magnesium source, when present, is selected from sodium and potassium gluconate, and when present in the cell-free protein synthesis system, the concentration of potassium glutamate is about 10 to 270 mM. When present in the cell-free protein synthesis system, the concentration of potassium gluconate is about 10 to 250 mM. When present, the magnesium source comprises magnesium glutamate. The concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM. When present, the phosphate source comprises potassium phosphate. The concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM, the buffer, if present, comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL, the cell-free protein synthesis system containing free amino acids endogenous to the lysed bacteria, the concentration of the free amino acids in the cell-free protein synthesis system being less than 2 mM, the cell-free protein synthesis system containing free nucleotides endogenous to the lysed bacteria, the concentration of the nucleotides in the cell-free protein synthesis system being 1 to 100 μg / mL, The concentration of ATP in the minimal medium is less than 1.5 mM, the cell-free protein synthesis system contains free NADH / NADPH inherent in lysed bacteria, the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, the carbon source being selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium being 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium being 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium being 0.05% to 5% (w / v).

[0078] In some embodiments, the phosphate source comprises a sodium phosphate buffer or a potassium phosphate buffer. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 5 to 250 mM. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 10 to 200 mM. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 15 to 150 mM. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 20 to 100 mM. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 25 to 75 mM. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 37.5 to 62.5 mM. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 45 to 55 mM. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 47.5 to 52.5 mM. In some embodiments, the concentration of the potassium phosphate buffer in the minimal medium is 50 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the phosphate source is selected from a sodium phosphate buffer and a potassium phosphate buffer, and the concentration of the potassium phosphate buffer in the minimal medium is 5 to 250 mM.

[0079] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L, the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L, the ammonium source, when present, comprises ammonium sulfate, the concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM, and the potassium ... ammonium sulfate. The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of the potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of the potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.the sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM; the buffer, if present, comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM; the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v); the concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL; the cell-free protein synthesis system containing free amino acids endogenous to lysed bacteria, the concentration of the free amino acids in the cell-free protein synthesis system being less than 2 mM; the cell-free protein synthesis system containing free nucleotides endogenous to lysed bacteria, the concentration of nucleotides in the cell-free protein synthesis system being less than 1.5 mM; the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), the phosphate source includes sodium phosphate buffer or potassium phosphate buffer, and the concentration of potassium phosphate in the minimal medium is 5 to 250 mM.

[0080] In some embodiments, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7. In some embodiments, the pH of the potassium phosphate buffer in the minimal medium is 6.8 to 7.6. In some embodiments, the pH of the potassium phosphate buffer in the minimal medium is 6.9 to 7.5. In some embodiments, the pH of the potassium phosphate buffer in the minimal medium is 7 to 7.4. In some embodiments, the pH of the potassium phosphate buffer in the minimal medium is 7.1 to 7.3. In some embodiments, the pH of the potassium phosphate buffer in the minimal medium is 7.2. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the phosphate source is selected from a sodium phosphate buffer and a potassium phosphate buffer, and the potassium phosphate buffer in the minimal medium has a pH of 6.7 to 7.7.

[0081] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0 to 40 g / L; the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L; the ammonium source, when present, comprises ammonium sulfate; the concentration of the ammonium source in the cell-free protein synthesis system is 2 to 20 mM; and the potassium source, when present, When present, the magnesium source is selected from potassium glutamate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM, the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM, the magnesium source, when present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM, and the phosphate source comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23 mM.the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL; the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria; the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM; the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria; the concentration of the nucleotides in the cell-free protein synthesis system is less than 1.5 mM; and the cell-free protein synthesis system contains free NADPH endogenous to the lysed bacteria. (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate; and the carbon source is D-glucose and glycerol. The minimal medium is selected from the group consisting of: a D-glucose concentration of 0.1% to 2.5% (w / v), a glycerol concentration of 0.25% to 25% (w / v), a sodium succinate concentration of 0.05% to 5% (w / v), a phosphate source comprising a sodium phosphate buffer or a potassium phosphate buffer, a potassium phosphate concentration of 5 to 250 mM, and a pH of the potassium phosphate buffer in the minimal medium of 6.7 to 7.7.

[0082] In some embodiments, the ammonium source comprises ammonium chloride. In some embodiments, the concentration of ammonium chloride in the minimal medium is 5 to 250 mM. In some embodiments, the concentration of ammonium chloride in the minimal medium is 10 to 200 mM. In some embodiments, the concentration of ammonium chloride in the minimal medium is 15 to 150 mM. In some embodiments, the concentration of ammonium chloride in the minimal medium is 20 to 100 mM. In some embodiments, the concentration of ammonium chloride in the minimal medium is 25 to 75 mM. In some embodiments, the concentration of ammonium chloride in the minimal medium is 37.5 to 62.5 mM. In some embodiments, the concentration of ammonium chloride in the minimal medium is 45 to 55 mM. In some embodiments, the concentration of ammonium chloride in the minimal medium is 47.5 to 52.5 mM. In some embodiments, the concentration of ammonium chloride in the minimal medium is 50 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the ammonium source comprises ammonium chloride, and the concentration of ammonium chloride in the minimal medium is 5 to 250 mM.

[0083] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7.8. , about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the cell-free protein synthesis system is maintained at a temperature of less than about 30°C, the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, the molecular crowder being selected from PEG 400 to 8000, maltodextrin, Ficoll 70 to 400, dextran, and serum albumin, optionally bovine serum albumin, When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L; when present, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; when present, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L; when present, the ammonium source comprises ammonium sulfate; when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2-20 mM; and when present, the potassium source is The cell-free protein synthesis system is selected from potassium glutamate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM, the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23 mM.the sulfur source comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM; the buffer comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM; the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v); the concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL; the cell-free protein synthesis system contains free amino acids present in the lysed bacteria, the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM; the cell-free protein synthesis system contains free nucleotides present in the lysed bacteria, the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM; the cell-free protein synthesis system contains free NADH / NADPH present in the lysed bacteria, and the concentration of the free NADH in the cell-free protein synthesis system is less than 1.5 mM. (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate; the carbon source is selected from D-glucose and glycerol, and the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v). The minimal medium contains a glycerol concentration of 0.25% to 25% (w / v), a sodium succinate concentration of 0.05% to 5% (w / v), a phosphate source containing a sodium phosphate buffer or a potassium phosphate buffer, a potassium phosphate concentration of 5 to 250 mM, a pH of the potassium phosphate buffer in the minimal medium of 6.7 to 7.7, an ammonium source containing ammonium chloride, and a ammonium chloride concentration of 5 to 250 mM.

[0084] In some embodiments, the sulfur source comprises sodium sulfate. In some embodiments, the concentration of sodium sulfate in the minimal medium is 0.5 to 25 mM. In some embodiments, the concentration of sodium sulfate in the minimal medium is 1 to 20 mM. In some embodiments, the concentration of sodium sulfate in the minimal medium is 1.5 to 15 mM. In some embodiments, the concentration of sodium sulfate in the minimal medium is 2 to 10 mM. In some embodiments, the concentration of sodium sulfate in the minimal medium is 2.5 to 7.5 mM. In some embodiments, the concentration of sodium sulfate in the minimal medium is 3.75 to 6.25 mM. In some embodiments, the concentration of sodium sulfate in the minimal medium is 4.5 to 5.5 mM. In some embodiments, the concentration of sodium sulfate in the minimal medium is 4.75 to 5.25 mM. In some embodiments, the concentration of sodium sulfate in the minimal medium is 5 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the sulfur source comprises sodium sulfate, and the concentration of sodium sulfate in the minimal medium is 0.5 to 25 mM.

[0085] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L, the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L, the ammonium source, when present, comprises ammonium sulfate, the concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM, and the potassium ... ammonium sulfate. The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of the potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of the potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.the sulfur source comprises ammonium sulfate, and the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM; the buffer comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, and the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM; the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v); and the concentration of DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL. The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cellular protein synthesis system does not contain tryptone, and the cell-free protein synthesis system does not contain yeast extract. The minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. The carbon source is selected from D-glucose and glycerol, and the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), and the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v). The minimal medium contains sodium succinate at a concentration of 0.05% to 5% (w / v), the phosphate source includes sodium phosphate buffer or potassium phosphate buffer, the potassium phosphate at a concentration of 5 to 250 mM, the potassium phosphate buffer in the minimal medium has a pH of 6.7 to 7.7, the ammonium source includes ammonium chloride, the ammonium chloride at a concentration of 5 to 250 mM, and the sulfur source includes sodium sulfate, the sodium sulfate at a concentration of 0.5 to 25 mM.

[0086] In some embodiments, the magnesium source comprises magnesium sulfate heptahydrate. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.2-20 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.3-19 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.4-18 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.5-17 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.6-16 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.7-15 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.8-14 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.9-13 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1-12 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.1-11 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.2-10 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.3-9 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.4-8 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.5-7 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.6-6 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.7-5 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.8-4 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.8 to 3 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.9 to 2.5 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.9 to 2.25 mM.In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 1.9 to 2.1 mM. In some embodiments, the concentration of magnesium sulfate heptahydrate in the minimal medium is 2 mM. Provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the magnesium source comprises magnesium sulfate heptahydrate, and the concentration of magnesium sulfate heptahydrate in the minimal medium is 0.2 to 20 mM.

[0087] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L, the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L, the ammonium source, when present, comprises ammonium sulfate, the concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM, and the potassium ... ammonium sulfate. The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of the potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of the potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.The sulfur source comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM, the buffer, when present, comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone. The synthetic system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is phosphate. The minimal medium contains a sodium phosphate buffer or a potassium phosphate buffer, the potassium phosphate concentration in the minimal medium is 5 to 250 mM, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7, the ammonium source contains ammonium chloride, the ammonium chloride concentration in the minimal medium is 5 to 250 mM, the sulfur source contains sodium sulfate, the sulfur source contains sodium sulfate, the sodium sulfate concentration in the minimal medium is 0.5 to 25 mM, and the magnesium source contains magnesium sulfate heptahydrate, the sodium sulfate heptahydrate concentration in the minimal medium is 0.It is 2 to 20 mM.

[0088] In some embodiments, the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source. In some embodiments, the iron source comprises iron citrate. In some embodiments, the concentration of iron citrate in the minimal medium is 10 to 1,000 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 20 to 900 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 30 to 800 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 40 to 700 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 50 to 600 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 60 to 500 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 70 to 400 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 80 to 300 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 90 to 200 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 90 to 150 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 95 to 125 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 95 to 105 μM. In some embodiments, the concentration of iron citrate in the minimal medium is 100 μM. (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate; wherein the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source; and wherein the iron source comprises iron citrate, and the concentration of the iron citrate in the minimal medium is 10 to 1,000 μM.

[0089] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0-40 g / L, the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L, the ammonium source, when present, comprises ammonium sulfate, the concentration of the ammonium source in the cell-free protein synthesis system is 2-20 mM, and the potassium ... ammonium sulfate. The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of the potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of the potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM, the buffer comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v), the concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL, The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone. The synthetic system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is phosphate. The minimal medium contains a sodium phosphate buffer or a potassium phosphate buffer, the potassium phosphate concentration in the minimal medium is 5 to 250 mM, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7, the ammonium source contains ammonium chloride, the ammonium chloride concentration in the minimal medium is 5 to 250 mM, the sulfur source contains sodium sulfate, the sulfur source contains sodium sulfate, the sodium sulfate concentration in the minimal medium is 0.5 to 25 mM, and the magnesium source contains magnesium sulfate heptahydrate, the sodium sulfate heptahydrate concentration in the minimal medium is 0.The trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, the iron source includes iron citrate, and the concentration of iron citrate in the minimal medium is 10 to 1,000 μM.

[0090] bacterial culture In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the bacteria is Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), or Vibrio natriegens (V. natriegens).

[0091] In some embodiments, bacteria are grown in any minimal medium. In some embodiments, bacteria are grown in lysogeny broth medium. In some embodiments, bacteria are pelleted (i.e., the supernatant is removed and the bacteria are allowed to settle), and the pellet is then resuspended in minimal medium at 4% (v / v), optionally further comprising about 12.5 μg / mL chloramphenicol and 50 μg / mL isopropyl β-d-1-thiogalactopyranoside (IPTG).

[0092] In some embodiments, bacteria are grown in minimal medium at 37°C and monitored until an OD600 of 7-10 is reached. In some embodiments, the bacteria are pelleted and the supernatant is discarded. In some embodiments, the bacteria are washed once with a buffer solution containing 10 mM Tris-acetate (pH 7.9), 14 mM magnesium glutamate, and 60 mM potassium glutamate at a ratio of 20 mL of buffer per gram of bacteria. In some embodiments, the bacteria are pelleted and resuspended in a buffer solution containing 1.1 mL of buffer per gram of bacteria to obtain a cell-buffer suspension. In some embodiments, the cell-buffer suspension is immersed in an ice-water bath for preparation of a cell-free cell extract.

[0093] cell-free cell extract In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the cell-free cell extract is prepared by preparing a suspension in 0.1 mL to 6.1 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in 0.2 mL to 5.6 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in 0.3 mL to 5.1 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in 0.4 mL to 4.6 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cellular extract is prepared by preparing a suspension in 0.5 mL to 4.1 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cellular extract is prepared by preparing a suspension in 0.6 mL to 3.6 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cellular extract is prepared by preparing a suspension in 0.7 mL to 3.1 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cellular extract is prepared by preparing a suspension in 0.8 mL to 2.6 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cellular extract is prepared by preparing a suspension in 0.9 mL to 2.1 mL of suspension buffer per gram of bacteria. In some embodiments, the cell-free cellular extract is prepared by preparing a suspension in 1 mL to 1.6 mL of suspension buffer per gram of bacteria.In some embodiments, the cell-free extract is prepared by preparing a suspension in which each gram of bacteria is suspended in 1.05 mL to 1.35 mL of suspension buffer. In some embodiments, the cell-free extract is prepared by preparing a suspension in which each gram of bacteria is suspended in 1.05 mL to 1.2 mL of suspension buffer.

[0094] In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of bacteria is suspended in 1.1 mL of suspension buffer. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when present in the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.(f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin, and the concentration of the molecular crowder, when present in the cell-free protein synthesis system, is about 0-40 g / L, and the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, and the cell-free protein synthesis system is maintained at a temperature of less than about 30°C. When present in the cell-free protein synthesis system, the concentration of the carbon source is about 0 to 50 g / L; the ammonium source, if present, comprises ammonium sulfate; the concentration of the ammonium source in the cell-free protein synthesis system is 2 to 20 mM; the potassium source, if present, is selected from potassium glutamate and potassium gluconate; the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; the magnesium source, if present, comprises magnesium glutamate; the concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM; the phosphate source comprises potassium phosphate; and the concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23 mM.The sulfur source comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM, the buffer, when present, comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone. The synthetic system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is phosphate. The minimal medium contains a sodium phosphate buffer or a potassium phosphate buffer, the potassium phosphate concentration in the minimal medium is 5 to 250 mM, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7, the ammonium source contains ammonium chloride, the ammonium chloride concentration in the minimal medium is 5 to 250 mM, the sulfur source contains sodium sulfate, the sulfur source contains sodium sulfate, the sodium sulfate concentration in the minimal medium is 0.5 to 25 mM, and the magnesium source contains magnesium sulfate heptahydrate, the sodium sulfate heptahydrate concentration in the minimal medium is 0.The trace mineral source is 2-20 mM, the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, the iron source includes iron citrate, and the concentration of iron citrate in the minimal medium is 10-1,000 μM. The cell-free cell extract is prepared by preparing a suspension in which 1 gram of bacteria is suspended in 0.1 mL-6.1 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by lysing the bacteria in the suspension. In some embodiments, the lysing comprises sonication. In some embodiments, the sonication is performed using a 10-second on / 10-second off cycle and 50% amplitude. In some embodiments, the sonication is performed until the input energy reaches approximately 3,500 joules. In some embodiments, the bacterial lysate is centrifuged. In some embodiments, the bacterial lysate is centrifuged at 12,000 RCF for 15 minutes at 4°C. In some embodiments, the supernatant is collected and immediately frozen at -80°C. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the cell-free cell extract is prepared by preparing a suspension in which each gram of bacteria is suspended in 0.1 mL to 6.1 mL of suspension buffer, and the cell-free cell extract is prepared by lysing the bacteria in the suspension. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the cell-free cell extract is present in a volume of 0.1 mL to 6 mL per gram of bacteria, respectively.A cell-free cell extract is prepared by lysing the bacteria in suspension in 1 mL of suspension buffer, where the lysis involves sonication.

[0095] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0 to 40 g / L, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, when present in the cell-free protein synthesis system, the concentration of the carbon source is about 0 to 50 g / L, the ammonium source, when present, comprises ammonium sulfate, when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM, and the potassium source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, when present in the cell-free protein synthesis system, the concentration of the carbon source is about 0 to 50 g / L, and the ammonium source, when present in the cell-free protein synthesis system, comprises ammonium sulfate, when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM, and the potassium source is glutamic acid. The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.The sulfur source comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM, the buffer, when present, comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone. The synthetic system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is phosphate. The minimal medium contains a sodium phosphate buffer or a potassium phosphate buffer, the potassium phosphate concentration in the minimal medium is 5 to 250 mM, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7, the ammonium source contains ammonium chloride, the ammonium chloride concentration in the minimal medium is 5 to 250 mM, the sulfur source contains sodium sulfate, the sulfur source contains sodium sulfate, the sodium sulfate concentration in the minimal medium is 0.5 to 25 mM, and the magnesium source contains magnesium sulfate heptahydrate, the sodium sulfate heptahydrate concentration in the minimal medium is 0.The concentration of the trace mineral source is 2 to 20 mM, the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, the iron source includes iron citrate, and the concentration of the iron citrate in the minimal medium is 10 to 1,000 μM, and the cell-free cell extract is prepared by preparing a suspension in which 1 gram of bacteria is suspended in 0.1 mL to 6.1 mL of suspension buffer, and the cell-free cell extract is prepared by lysing the bacteria in the suspension, the lysis including sonication.

[0096] In some embodiments, the suspension buffer comprises one or more of (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate. In some embodiments, the suspension buffer comprises one or more of (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the cell-free cell extract is prepared by preparing a suspension in which the cell-free cell extract is suspended in 0.1 mL to 6.1 mL of suspension buffer per gram of bacteria, wherein the suspension buffer comprises one or more of (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate.

[0097] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. The concentration of the molecular crowder in the cell-free protein synthesis system is about 0 to 40 g / L, the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L, the ammonium source, when present, comprises ammonium sulfate, the concentration of the ammonium source in the cell-free protein synthesis system is 2 to 20 mM, and the potassium ... about 0 to 20 mM, and the potassium source, when present, is selected from glucose, fructose, galactose, The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM, the buffer comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v), the concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL, The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone. The synthetic system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is phosphate. The minimal medium contains a sodium phosphate buffer or a potassium phosphate buffer, the potassium phosphate concentration in the minimal medium is 5 to 250 mM, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7, the ammonium source contains ammonium chloride, the ammonium chloride concentration in the minimal medium is 5 to 250 mM, the sulfur source contains sodium sulfate, the sulfur source contains sodium sulfate, the sodium sulfate concentration in the minimal medium is 0.5 to 25 mM, and the magnesium source contains magnesium sulfate heptahydrate, the sodium sulfate heptahydrate concentration in the minimal medium is 0.The trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, the iron source includes iron citrate, and the concentration of the iron citrate in the minimal medium is 10 to 1,000 μM. The cell-free cell extract is prepared by preparing a suspension in which 1 gram of bacteria is suspended in 0.1 mL to 6.1 mL of a suspension buffer, and the cell-free cell extract is prepared by lysing the bacteria in the suspension, the lysing including sonication, and the suspension buffer including one or more of (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate.

[0098] In some embodiments, the concentration of Tris-acetate in the suspension buffer is 1 to 100 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 2 to 90 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 3 to 80 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 4 to 70 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 5 to 60 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 6 to 50 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 7 to 40 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 8 to 30 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 9 to 20 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 9 to 15 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 9 to 12.5 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 9 to 11 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 10 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the cell-free cell extract is prepared by preparing a suspension in which the bacteria are suspended in 0.1 mL to 6.1 mL of suspension buffer per gram of bacteria, wherein the suspension buffer comprises one or more of (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, and the concentration of Tris-acetate in the suspension buffer is 1 to 100 mM.

[0099] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. The concentration of the molecular crowder in the cell-free protein synthesis system is about 0 to 40 g / L, the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L, the ammonium source, when present, comprises ammonium sulfate, the concentration of the ammonium source in the cell-free protein synthesis system is 2 to 20 mM, and the potassium ... about 0 to 20 mM, and the potassium source, when present, is selected from glucose, fructose, galactose, The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM, the buffer comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v), the concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL, The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone. The synthetic system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is phosphate. The minimal medium contains a sodium phosphate buffer or a potassium phosphate buffer, the potassium phosphate concentration in the minimal medium is 5 to 250 mM, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7, the ammonium source contains ammonium chloride, the ammonium chloride concentration in the minimal medium is 5 to 250 mM, the sulfur source contains sodium sulfate, the sulfur source contains sodium sulfate, the sodium sulfate concentration in the minimal medium is 0.5 to 25 mM, and the magnesium source contains magnesium sulfate heptahydrate, the sodium sulfate heptahydrate concentration in the minimal medium is 0.The trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, the iron source includes iron citrate, and the concentration of the iron citrate in the minimal medium is 10 to 1,000 μM, the cell-free cell extract is prepared by preparing a suspension in which 1 gram of bacteria is suspended in 0.1 mL to 6.1 mL of a suspension buffer, and the cell-free cell extract is prepared by lysing the bacteria in the suspension, the lysis including sonication, and the suspension buffer includes one or more of (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, and the concentration of Tris-acetate in the suspension is 1 to 100 mM.

[0100] In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 1 to 100 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 2 to 90 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 3 to 80 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 4 to 70 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 5 to 60 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 6 to 50 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 7 to 40 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 8 to 30 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 9 to 20 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 10 to 19 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 11-18 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 12-17 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 13-16 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 13-15 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 14 mM.In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the cell-free cell extract is prepared by preparing a suspension in which the bacteria are suspended in 0.1 mL to 6.1 mL of a suspension buffer per gram of bacteria, wherein the suspension buffer comprises one or more of: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, and the concentration of the magnesium glutamate in the suspension buffer is 1 to 100 mM.

[0101] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7.9. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; and the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin. When present in the cell-free protein synthesis system, the concentration of the molecular crowder is about 0 to 40 g / L, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L, the ammonium source, when present, comprises ammonium sulfate, the concentration of the ammonium source in the cell-free protein synthesis system is 2 to 20 mM, and the potassium source, when present, is glutathione. The cell-free protein synthesis system is provided with a magnesium source selected from potassium glutamate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system being about 10 to 270 mM, the concentration of potassium gluconate in the cell-free protein synthesis system being about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system being 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system being about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM, the buffer comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v), the concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL, The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone. The synthetic system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is phosphate. The minimal medium contains a sodium phosphate buffer or a potassium phosphate buffer, the potassium phosphate concentration in the minimal medium is 5 to 250 mM, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7, the ammonium source contains ammonium chloride, the ammonium chloride concentration in the minimal medium is 5 to 250 mM, the sulfur source contains sodium sulfate, the sulfur source contains sodium sulfate, the sodium sulfate concentration in the minimal medium is 0.5 to 25 mM, and the magnesium source contains magnesium sulfate heptahydrate, the sodium sulfate heptahydrate concentration in the minimal medium is 0.The trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, the iron source includes iron citrate, and the concentration of the iron citrate in the minimal medium is 10 to 1,000 μM, the cell-free cell extract is prepared by preparing a suspension in which 1 gram of bacteria is suspended in 0.1 mL to 6.1 mL of a suspension buffer, and the cell-free cell extract is prepared by lysing the bacteria in the suspension, the lysing including sonication, and the suspension buffer includes one or more of (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, the concentration of Tris-acetate in the suspension is 1 to 100 mM, and the concentration of magnesium glutamate in the suspension buffer is 1 to 100 mM.

[0102] In some embodiments, the concentration of potassium glutamate in the suspension buffer is 1 to 180 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 5 to 170 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 10 to 160 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 15 to 150 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 20 to 140 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 25 to 130 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 30 to 120 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 35 to 110 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 40 to 100 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 45 to 90 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 50 to 80 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 55 to 70 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 55 to 65 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 57.5 to 62.5 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 60 mM.In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the cell-free cell extract is prepared by preparing a suspension in which the bacteria are suspended in 0.1 mL to 6.1 mL of a suspension buffer per gram of bacteria, wherein the suspension buffer comprises one or more of: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, wherein the concentration of potassium glutamate in the suspension buffer is 1 to 180 mM.

[0103] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria, and the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, or about 7.2 to 7.8. , about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the cell-free protein synthesis system is maintained at a temperature of less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, when present, is selected from PEG 400 to 8000, maltodextrin, Ficoll 70 to 400, dextran, and serum albumin, optionally bovine serum albumin. The concentration of the molecular crowder in the cell-free protein synthesis system is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, if present, and the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L. The ammonium source, if present, comprises ammonium sulfate. The concentration of the ammonium source in the cell-free protein synthesis system is 2 to 20 mM. The potassium source, if present, The cell-free protein synthesis system is selected from potassium glutamate and potassium gluconate, the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM, the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM, the magnesium source, if present, comprises magnesium glutamate, the concentration of the magnesium source in the cell-free protein synthesis system is 0 to about 10 mM, and the phosphate source, if present, comprises potassium phosphate, the concentration of the phosphate source in the cell-free protein synthesis system is about 5 to about 23 mM.The sulfur source, if present, comprises ammonium sulfate, the concentration of the sulfur source in the cell-free protein synthesis system being 2 to 20 mM, the buffer comprises a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, the concentration of the buffer in the cell-free protein synthesis system being 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system being 10% to 70% (v / v), the concentration of DNA or RNA encoding a protein of interest in the cell-free protein synthesis system being 1 to 100 μg / mL, The cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, and the concentration of nucleotides in the cell-free protein synthesis system is less than 1.5 mM. The cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, and the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone. The synthetic system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is phosphate. The minimal medium contains a sodium phosphate buffer or a potassium phosphate buffer, the potassium phosphate concentration in the minimal medium is 5 to 250 mM, the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7, the ammonium source contains ammonium chloride, the ammonium chloride concentration in the minimal medium is 5 to 250 mM, the sulfur source contains sodium sulfate, the sulfur source contains sodium sulfate, the sodium sulfate concentration in the minimal medium is 0.5 to 25 mM, and the magnesium source contains magnesium sulfate heptahydrate, the sodium sulfate heptahydrate concentration in the minimal medium is 0.The trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, the iron source includes iron citrate, and the concentration of the iron citrate in the minimal medium is 10 to 1,000 μM. The cell-free cell extract is prepared by preparing a suspension in which 1 gram of bacteria is suspended in 0.1 mL to 6.1 mL of a suspension buffer, and the cell-free cell extract is prepared by lysing the bacteria in the suspension, the lysis including sonication. The suspension buffer includes one or more of (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, the concentration of Tris-acetate in the suspension is 1 to 100 mM, the concentration of magnesium glutamate in the suspension buffer is 1 to 100 mM, and the concentration of potassium glutamate in the suspension buffer is 1 to 180 mM.

[0104] Cell-free protein synthesis In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising using any one of the cell-free protein synthesis systems disclosed herein. In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the protein of interest when maintained at that temperature. In some embodiments, the method comprises (3) purifying the protein of interest.

[0105] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at that temperature; wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5.

[0106] In some embodiments, provided herein are methods for preparing a protein of interest, the methods comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the protein of interest when maintained at the temperature;

[0107] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature, wherein the cell-free protein synthesis system has a pH of about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, and the temperature is less than about 30°C.

[0108] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature, wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0109] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0110] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin.

[0111] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system produces the protein of interest when maintained at that temperature. The pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, and the temperature is less than about 30°C. The reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder is selected from PEG 400 to 8000, maltodextrin, Ficoll 70 to 400, dextran, and serum albumin, optionally bovine serum albumin.

[0112] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the concentration of the molecular crowder in the cell-free protein synthesis system is about 0 to 40 g / L.

[0113] In some embodiments, provided herein are methods for preparing a protein of interest, the methods comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system produces the protein of interest when maintained at that temperature; wherein the pH of the cell-free protein synthesis system is about (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution; wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; and the concentration of the molecular crowder in the cell-free protein synthesis system is about 0-40 g / L.

[0114] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose.

[0115] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system produces the protein of interest when maintained at that temperature; wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin, and the concentration of the molecular crowder in the cell-free protein synthesis system is about 0-40 g / L. The carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose.

[0116] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L.

[0117] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, and the temperature is less than about 30° C., and the reaction mixture is free of free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria. (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin, and the concentration of the molecular crowder in the cell-free protein synthesis system is about 0-40 g / L, and the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, and the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L.

[0118] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the concentration of the ammonium source in the cell-free protein synthesis system is about 2 to 20 mM.

[0119] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system produces the protein of interest when maintained at the temperature; wherein the cell-free protein synthesis system has a pH of about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30° C., and the reaction mixture is further treated with (a) a molecular crowder. (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin, and the concentration of the molecular crowder in the cell-free protein synthesis system, if present, is about 0-40 g / L, and the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, and the concentration of the carbon source, if present, in the cell-free protein synthesis system is about 0-50 g / L, and the concentration of the ammonium source, if present in the cell-free protein synthesis system, is 2-20 mM.

[0120] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the potassium source is selected from potassium glutamate and potassium gluconate.

[0121] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature, wherein the cell-free protein synthesis system has a pH of about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture comprises: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a soluble ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder, if present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin, and the concentration of the molecular crowder in the cell-free protein synthesis system, if present, is about 0-40 g / L, and the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, and the concentration of the carbon source, if present in the cell-free protein synthesis system, is about 0-50 g / L, and the concentration of the ammonium source, if present in the cell-free protein synthesis system, is 2-20 mM, and the potassium source is selected from potassium glutamate and potassium gluconate.

[0122] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer; and wherein the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM.

[0123] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system produces the protein of interest when maintained at the temperature, the cell-free protein synthesis system having a pH of about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature being less than about 30° C., and the reaction mixture comprising: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a maltodextrin; the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; the concentration of the molecular crowder in the cell-free protein synthesis system is about 0-40 g / L; the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L; the concentration of the ammonium source, when present in the cell-free protein synthesis system, is 2-20 mM; and the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of potassium glutamate in the cell-free protein synthesis system is about 10-270 mM.

[0124] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer; and the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM.

[0125] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30° C., and the reaction mixture comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer. the molecular crowder, if present, is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; the concentration of the molecular crowder in the cell-free protein synthesis system is about 0-40 g / L; the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L and, if present in the cell-free protein synthesis system, is 2-20 mM; and the potassium source, if present, is selected from potassium glutamate and potassium gluconate; the concentration of potassium glutamate in the cell-free protein synthesis system is about 10-270 mM, and the concentration of potassium gluconate in the cell-free protein synthesis system is about 10-250 mM.

[0126] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the concentration of the magnesium source in the cell-free protein synthesis system is about 0 to 10 mM.

[0127] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30° C., the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria. and producing a protein of interest when the cell-free protein synthesis system is maintained at a temperature of about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture contains at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer. The method further comprises the steps of: when the molecular crowder is present, the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran, and serum albumin, optionally bovine serum albumin; the concentration of the molecular crowder in the cell-free protein synthesis system is about 0-40 g / L; and the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; and the concentration of the carbon source in the cell-free protein synthesis system is about 0-40 g / L. The concentration of the ammonium source, if present in the cell-free protein synthesis system, is 2 to 20 mM; the potassium source, if present, is selected from potassium glutamate and potassium gluconate; the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; and the concentration of the magnesium source, if present in the cell-free protein synthesis system, is 0 to about 10 mM.

[0128] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the concentration of the phosphate source in the cell-free protein synthesis system, if present, is from about 5 to about 23.5 mM.

[0129] In some embodiments, a method for preparing a protein of interest is described herein, comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C. (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the cell-free protein synthesis system has a pH of about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, and the temperature is less than about 30°C. The reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder is selected from the group consisting of PEG 400 to 8000, maltodextrin, and Ficoll. The concentration of the molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, when present in the cell-free protein synthesis system, is about 0 to 40 g / L, and the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, when present in the cell-free protein synthesis system, is about 0 to 50 g / L. The concentration of the potassium source in the cell-free protein synthesis system is 2 to 20 mM, the potassium source, when present, is selected from potassium glutamate and potassium gluconate, the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM, the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM, the concentration of the magnesium source, when present in the cell-free protein synthesis system, is 0 to about 10 mM, and the concentration of the phosphate source, when present in the cell-free protein synthesis system, is about 5 to about 23.5 mM.

[0130] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the concentration of the sulfur source in the cell-free protein synthesis system is about 2 to 20 mM.

[0131] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., the system producing the protein of interest when maintained at that temperature. and (g) a sulfur source; and (h) a buffer solution, wherein the cell-free protein synthesis system has a pH of about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, and the temperature is less than about 30°C. The reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder, when present, is selected from the group consisting of PEG 400-8000, maltodextrin, Fi The concentration of the molecular crowder, when present in the cell-free protein synthesis system, is selected from coll70-400, dextran, and serum albumin, optionally bovine serum albumin; the concentration of the molecular crowder, when present in the cell-free protein synthesis system, is about 0-40 g / L; the carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source in the cell-free protein synthesis system is about 0-50 g / L; the concentration of the ammonium source, when present in the cell-free protein synthesis system, is 2-20 mM; When present, the sodium source is selected from potassium glutamate and potassium gluconate, the potassium glutamate concentration in the cell-free protein synthesis system is about 10 to 270 mM, the potassium gluconate concentration in the cell-free protein synthesis system is about 10 to 250 mM, the magnesium source concentration, when present in the cell-free protein synthesis system, is 0 to about 10 mM, the phosphate source concentration, when present in the cell-free protein synthesis system, is about 5 to about 23.5 mM, and the sulfur source concentration, when present in the cell-free protein synthesis system, is 2 to 20 mM.

[0132] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature; wherein the reaction mixture further comprises at least one of: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the concentration of the buffer solution in the cell-free protein synthesis system is 50 to 300 mM.

[0133] In some embodiments, provided herein are methods for preparing a protein of interest, the methods comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the pH of the cell-free protein synthesis system is , about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer solution, wherein the molecular crowder is selected from PEG 400 to 8000, maltodextrin, Ficoll 70 to 400, dextran, and serum albumin, optionally bovine serum albumin. When present in the cell-free protein synthesis system, the concentration of the molecular crowder, if present, is about 0 to 40 g / L; the carbon source, if present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source in the cell-free protein synthesis system is about 0 to 50 g / L; when present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM; and the potassium source, if present, is selected from potassium glutamate and potassium gluconate. The selected cell-free protein synthesis system has a potassium glutamate concentration of about 10 to 270 mM, a potassium gluconate concentration of about 10 to 250 mM, a magnesium source concentration of 0 to about 10 mM when present in the cell-free protein synthesis system, a phosphate source concentration of about 5 to about 23.5 mM when present in the cell-free protein synthesis system, a sulfur source concentration of 2 to 20 mM when present in the cell-free protein synthesis system, and a buffer concentration of 50 to 300 mM when present in the cell-free protein synthesis system.

[0134] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at that temperature; wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v).

[0135] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH when maintained at the temperature. and producing a protein of interest, wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEFC. The molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present in the cell-free protein synthesis system ... When present in the synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 50-300 mM, and the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10%-70% (v / v).

[0136] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at that temperature; wherein the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL.

[0137] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain DNA or RNA encoding the protein of interest when maintained at that temperature. and producing a protein of formula (I), wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEG-4. the concentration of the molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source ... When present in the synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 5 mM, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), and the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL.

[0138] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system produces the protein of interest when maintained at that temperature; wherein the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM.

[0139] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH when maintained at the temperature. and producing a protein of interest, wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEFC. The molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present in the cell-free protein synthesis system ... When present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, and the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM.

[0140] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system is free of free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature less than about 30° C., wherein the system is free of free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature, wherein the system produces the protein of interest when maintained at the temperature; wherein the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.5 mM.

[0141] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH when maintained at the temperature. and producing a protein of interest, wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEFC. The molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present in the cell-free protein synthesis system ... When present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 5 mM, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, and the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM.

[0142] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system produces the protein of interest when maintained at that temperature; wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM.

[0143] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain DNA or RNA encoding the protein of interest when maintained at that temperature. and producing a protein of formula (I), wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEG-4. the concentration of the molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source ... When present in the synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 5 mM, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, and the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM.

[0144] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system produces the protein of interest when maintained at that temperature; wherein the cell-free protein synthesis system does not contain tryptone. In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) obtaining a cell-free protein synthesis system, the system comprising (a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium, and (b) a reaction mixture comprising DNA or RNA encoding the protein of interest, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature, wherein the system produces the protein of interest when maintained at the temperature, the cell-free protein synthesis system being free of yeast extract.

[0145] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH when maintained at the temperature. and producing a protein of interest, wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEFC. The molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present in the cell-free protein synthesis system ... When present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 5 mM, and if present, the concentration of the sulfur source is 2 to 20 mM. The concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM. The concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v). The concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL. The concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. The concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. The cell-free protein synthesis system does not contain tryptone, and the cell-free protein synthesis system does not contain yeast extract.

[0146] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature, wherein the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate.

[0147] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH when maintained at the temperature. and producing a protein of interest, wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEFC. The molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present in the cell-free protein synthesis system ... When present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the cell-free protein synthesis system is 5 mM, and if present in the cell-free protein synthesis system, the concentration of the sulfur source is 2 to 20 mM, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, and the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate.

[0148] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature, wherein the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, and the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v).

[0149] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain DNA or RNA encoding the protein of interest when maintained at that temperature. and producing a protein of formula (I), wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEG-4. the concentration of the molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source ... When present in the synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.When present in the cell-free protein synthesis system, the concentration of the sulfur source is 2 to 20 mM, the concentration of the buffer solution in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, The concentration of free NADH / NADPH in the minimal medium is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source, and (g) sodium succinate, the carbon source is selected from D-glucose and glycerol, and the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v).

[0150] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature, wherein the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, and the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v).

[0151] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH when maintained at the temperature. and producing a protein of interest, wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEFC. The molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present in the cell-free protein synthesis system ... When present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.When present in the cell-free protein synthesis system, the concentration of the sulfur source is 2 to 20 mM, the concentration of the buffer solution in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, and the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source, and (g) sodium succinate, the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), and the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v).

[0152] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v).

[0153] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain DNA or RNA encoding the protein of interest when maintained at that temperature. and producing a protein of formula (I), wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEG-4. the concentration of the molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source ... When present in the synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.When present in the cell-free protein synthesis system, the concentration of the sulfur source is 2 to 20 mM, the concentration of the buffer solution in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, The protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, the minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source, and (g) sodium succinate, the carbon source being selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium being 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium being 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium being 0.05% to 5% (w / v).

[0154] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the minimal medium comprises at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the phosphate source comprises a sodium phosphate buffer or a potassium phosphate buffer, and wherein the concentration of potassium phosphate in the minimal medium is 5 to 250 mM.

[0155] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH when maintained at the temperature. and producing a protein of interest, wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEFC. The molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. The carbon source, when present in the cell-free protein synthesis system ... When present in the cell-free protein synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of the potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of the potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.the concentration of the cell-free protein synthesis system is 50 to 300 mM; the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v); the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL; the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM; the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM; the cell-free protein synthesis system does not contain tryptone; The extract-free minimal medium contains at least one of (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), and the phosphate source is sodium phosphate buffer or potassium phosphate buffer, and the concentration of the potassium phosphate buffer in the minimal medium is 5 to 250 mM.

[0156] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at the temperature, the minimal medium comprising at least one of: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the phosphate source comprises a sodium phosphate buffer or a potassium phosphate buffer, and wherein the potassium phosphate in the minimal medium has a pH of 6.7 to 7.7.

[0157] In some embodiments, a method for preparing a protein of interest comprises: (1) incubating a reaction mixture containing DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except as endogenous to the lysed bacteria; and (2) maintaining the system at a temperature of less than about 30° C., wherein the system does not contain DNA or RNA encoding the protein of interest when maintained at that temperature. and producing a protein of formula (I), wherein the pH of the cell-free protein synthesis system is about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, the temperature is less than about 30°C, and the reaction mixture further comprises at least one of (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder, if present, is PEG-4. the concentration of the molecular crowder, when present in the cell-free protein synthesis system, is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose; the concentration of the carbon source ... When present in the synthesis system, the concentration of the ammonium source is 2 to 20 mM; when present, the potassium source is selected from potassium glutamate and potassium gluconate; the concentration of potassium glutamate in the cell-free protein synthesis system is about 10 to 270 mM; the concentration of potassium gluconate in the cell-free protein synthesis system is about 10 to 250 mM; when present in the cell-free protein synthesis system, the concentration of the magnesium source is 0 to about 10 mM; and when present in the cell-free protein synthesis system, the concentration of the phosphate source is about 5 to about 23 mM.The concentration of the sulfur source in the cell-free protein synthesis system is 2 to 20 mM, the concentration of the buffer in the cell-free protein synthesis system is 50 to 300 mM, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 10% to 70% (v / v), the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is 1 to 100 μg / mL, the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, the cell-free protein synthesis system does not contain tryptone, the cell-free protein synthesis system does not contain yeast extract, and the minimal medium is (a (b) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) at least one of sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, the concentration of D-glucose in the minimal medium is 0.1% to 2.5% (w / v), the concentration of glycerol in the minimal medium is 0.25% to 25% (w / v), and the concentration of sodium succinate in the minimal medium is 0.05% to 5% (w / v), the phosphate source is a sodium phosphate buffer or a potassium phosphate buffer, the concentration of the potassium phosphate buffer in the minimal medium is 5 to 250 mM, and the pH of the potassium phosphate buffer in the minimal medium is 6.7 to 7.7.

[0158] In some embodiments, provided herein are methods for preparing a protein of interest, the method comprising: (1) incubating a reaction mixture comprising DNA or RNA encoding the protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; and (2) maintaining the system at a t...

Claims

1. A cell-free protein synthesis system, the system comprising: a) a cell-free cell extract comprising supernatant recovered from lysed bacteria grown in minimal medium; b) a reaction mixture containing DNA or RNA encoding a protein of interest; Including, the system does not contain free amino acids, free nucleotides or free NADH / NADPH except when endogenous to the lysed bacteria; Cell-free protein synthesis system.

2. i. the bacterium is E. coli, B. subtilis, or V. natriegens; and / or ii. the pH of the system is from about 7.0 to about 8.0, optionally 7.5; and / or iii. The system is maintained at a temperature of about 20°C to about 30°C, optionally about 25°C; The system of claim 1.

3. the reaction mixture a) molecular crowder; b) carbon source; c) an ammonium source; d) a potassium source; e) a magnesium source; f) a phosphate source; and g) a sulfur source; and optionally h) buffer solution 3. The system of claim 1 or claim 2, further comprising:

4. i. the molecular crowder is selected from PEG 400-8000, maltodextrin, Ficoll 70-400, dextran and bovine serum albumin, optionally PEG 8000; and / or ii. the concentration of the molecular crowder in the system is between 0 and 40 g / L, optionally 20 g / L; and / or iii. The carbon source in the reaction mixture comprises a sugar; and / or iv. the carbon source in the reaction mixture comprises maltose, and / or v. the carbon source in the reaction mixture does not contain maltose, and / or vi. the concentration of the carbon source in the system is 0 to 50 g / L, optionally 20 g / L; and / or vii. the ammonium source in the reaction mixture comprises ammonium sulfate, and / or viii. the concentration of the ammonium source in the system is 2 to 20 mM, optionally 12 mM; and / or ix. the potassium source in the reaction mixture is selected from potassium glutamate and potassium gluconate, and / or x. the potassium source in the reaction mixture is potassium glutamate, and the concentration of potassium glutamate in the system is 10 to 300 mM, optionally 20 mM; and / or xi. The potassium source in the reaction mixture is potassium gluconate, and the concentration of potassium gluconate in the system is 10 to 250 mM, optionally 100 mM; and / or xii. the magnesium source in the reaction mixture comprises magnesium glutamate, and / or xiii. the concentration of the magnesium source in the system is 0-10 mM, optionally 4 mM; and / or xiv. the phosphate source in the reaction mixture comprises potassium phosphate; and / or xv. the concentration of the phosphate source in the system is 5 to 25 mM, optionally 13 mM; and / or xvi. the sulfur source in the reaction mixture comprises ammonium sulfate, and / or xvii. The concentration of the sulfur source in the system is 2 to 20 mM, optionally 12 mM; and / or xviii. the system comprises the buffer in the reaction mixture, and the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer; and / or xix. The concentration of the buffer in the system is 50 to 300 mM, optionally 100 mM; The system of claim 3.

5. The minimal medium is a) carbon source; b) an ammonium source; c) a magnesium source; d) phosphate source; e) a sulfur source; and f) a trace mineral source; and optionally g) Sodium succinate Including, Optionally, i. the carbon source in the minimal medium is selected from D-glucose and glycerol, and / or ii. The concentration of the carbon source in the minimal medium is 0.5% to 4% (w / v), and / or iii. The concentration of glycerol in the minimal medium is 2.5% (w / v), and / or iv. the ammonium source in the minimal medium comprises ammonium chloride, and / or v. the concentration of the ammonium source in the minimal medium is 50 mM, and / or vi. the magnesium source in the minimal medium comprises magnesium sulfate heptahydrate, and / or vii. The concentration of the magnesium source in the minimal medium is 2 mM; and / or viii. The phosphate source in the minimal medium comprises a sodium phosphate buffer or a potassium phosphate buffer; and / or ix. The concentration of the phosphate source in the minimal medium is 25 mM to 150 mM; and / or x. The concentration of the phosphate source in the minimal medium is 50 mM, and / or xi. The pH of the sodium phosphate buffer or the potassium phosphate buffer in the minimal medium is 7.2, and / or xii. The sulfur source in the minimal medium comprises sodium sulfate, and / or xiii. The concentration of the sulfur source in the minimal medium is 5 mM; and / or xiv. the trace mineral source in the minimal medium is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source; and / or xv. the iron source comprises iron citrate, and / or xvi. The concentration of the iron source in the minimal medium is 100 μM, and / or xvii. The minimal medium contains sodium succinate, and the concentration of the sodium succinate in the minimal medium is 0.375% (w / v); A system according to any one of claims 1 to 4.

6. A method for preparing a protein of interest, comprising using a system according to any one of claims 1 to 5.

7. A reaction mixture for use in a cell-free protein synthesis system, the reaction mixture comprising DNA or RNA encoding a protein of interest; a) molecular crowder; b) carbon source; c) an ammonium source; d) a potassium source; e) a magnesium source; f) a phosphate source; and g) a sulfur source; and optionally h) a buffer; and optionally i) Sodium chloride Further comprising: Optionally, one or more of components (a) through (h) are as defined in claim 4.

8. 1. A method for preparing a protein of interest, comprising: 1) incubating a reaction mixture containing DNA or RNA encoding a protein of interest with a cell-free cell extract comprising supernatant collected from lysed bacteria grown in minimal medium, the system being free of free amino acids, free nucleotides, or free NADH / NADPH except when endogenous to the lysed bacteria; 2) maintaining the system at a temperature of less than about 30°C, wherein the system produces the protein of interest when maintained at said temperature.

9. 9. The method of claim 8, further comprising purifying the protein of interest.

10. 10. The method of claim 8 or claim 9, wherein the bacterium is E. coli, B. subtilis, or V. natriegens.

11. the reaction mixture a) molecular crowder; b) carbon source; c) an ammonium source; d) a potassium source; e) a magnesium source; f) a phosphate source; and g) a sulfur source; and optionally h) buffer solution Further comprising:

11. The method of any one of claims 8 to 10, wherein optionally one or more of components (a) to (h) are as defined in claim 4.

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