Figure 1. A comparison of cell-free and in vivo protein synthesis methods. Through visualization of the main steps of in vitro and in vivo protein expression, the advantages of cell-free protein synthesis emerge. These include the elimination of the transformation step, an open reaction for direct manipulation of the environment of protein production, the lack of constraints based on the cell’s life objectives, the channeling of all energy toward production of the protein of interest, and the ability to store extracts for on-demand protein expression. Green cylinders represent synthesized green fluorescent protein (GFP).

Figure 1. A comparison of cell-free and in vivo protein synthesis methods. Through visualization of the main steps of in vitro and in vivo protein expression, the advantages of cell-free protein synthesis emerge. These include the elimination of the transformation step, an open reaction for direct manipulation of the environment of protein production, the lack of constraints based on the cell’s life objectives, the channeling of all energy toward production of the protein of interest, and the ability to store extracts for on-demand protein expression. Green cylinders represent synthesized green fluorescent protein (GFP).

Figure 2. Comparison of protein yields across cell-free platforms. The volumetric yield of each platform is reported for batch reactions producing GFP. Platforms that report volumetric yield for reporter proteins luciferase (*) or ChiAΔ4 (**) are indicated. Information for batch mode protein yields of the Arabidopsis and Neurospora crassa platforms was not found. Yields were obtained from the following sources: E. coli [8] , wheat germ [ Vibrio natriegens [ Leishmania tarentolae [ Pseudomonas putida [ Streptomyces [ Bacillus megaterium [ Bacillus subtilis [ Comparison of protein yields across cell-free platforms. The volumetric yield of each platform is reported for batch reactions producing GFP. Platforms that report volumetric yield for reporter proteins luciferase (*) or ChiAΔ4 (**) are indicated. Information for batch mode protein yields of theandplatforms was not found. Yields were obtained from the following sources:wheat germ [ 9 ], 10 ], 11 ], tobacco [ 12 ], HeLa [ 13 ], 14 ], 15 ], 16 ], Chinese hamster ovary [ 17 ], insect [ 18 ], 16 ], yeast [ 19 ], archaeal [ 20 ], and rabbit reticulocyte [ 21 ].

Figure 2. Comparison of protein yields across cell-free platforms. The volumetric yield of each platform is reported for batch reactions producing GFP. Platforms that report volumetric yield for reporter proteins luciferase (*) or ChiAΔ4 (**) are indicated. Information for batch mode protein yields of the Arabidopsis and Neurospora crassa platforms was not found. Yields were obtained from the following sources: E. coli [8] , wheat germ [ Vibrio natriegens [ Leishmania tarentolae [ Pseudomonas putida [ Streptomyces [ Bacillus megaterium [ Bacillus subtilis [ Comparison of protein yields across cell-free platforms. The volumetric yield of each platform is reported for batch reactions producing GFP. Platforms that report volumetric yield for reporter proteins luciferase (*) or ChiAΔ4 (**) are indicated. Information for batch mode protein yields of theandplatforms was not found. Yields were obtained from the following sources:wheat germ [ 9 ], 10 ], 11 ], tobacco [ 12 ], HeLa [ 13 ], 14 ], 15 ], 16 ], Chinese hamster ovary [ 17 ], insect [ 18 ], 16 ], yeast [ 19 ], archaeal [ 20 ], and rabbit reticulocyte [ 21 ].

Figure 3. Comparison of batch, continuous flow, and continuous exchange reaction formats. Batch reactions contain all the necessary reactants within a single reaction vessel. Continuous exchange formats utilize a dialysis membrane that allows reactants to move into the reaction and byproducts to move out, while the protein of interest remains in the reaction compartment. Continuous flow formats allow a feed solution to be continuously pumped into the reaction chamber while the protein of interest and other reaction byproducts are filtered out of the reaction.

Figure 3. Comparison of batch, continuous flow, and continuous exchange reaction formats. Batch reactions contain all the necessary reactants within a single reaction vessel. Continuous exchange formats utilize a dialysis membrane that allows reactants to move into the reaction and byproducts to move out, while the protein of interest remains in the reaction compartment. Continuous flow formats allow a feed solution to be continuously pumped into the reaction chamber while the protein of interest and other reaction byproducts are filtered out of the reaction.

Figure 4. High adoption cell-free platforms and their applications. (A) Web of the applications enabled by high adoption cell-free platforms. The connections shown are based on applications that have been published for each respective platform. Applications under “difficult to synthesize proteins” include the production of antibodies, large proteins, ice structuring proteins, and metalloproteins. Miscellaneous applications include studies of translational machinery, genetic circuits, metabolic engineering, and genetic code expansion. (B) Cumulative number of peer-reviewed publications over the last 60 years for high adoption platforms. The metric of cumulative publications by platform is used to indicate which platforms are most utilized, with platforms having over 25 papers categorized as high adoption. These data were generated by totaling papers from a PubMed Boolean search of the following: (“cell free protein synthesis” OR “in vitro transcription translation” OR “in vitro protein synthesis” OR “cell free protein expression” OR “tx tl” OR “cell-free translation”) AND “platform name.” The platform name used for each search corresponds to the name listed in the graph’s key. This information was collected on 23 December 2018, and the search results for each platform can be found in Supplemental Information. While this metric is an indicator of the level of adoption for each platform, it does suffer from false positive search results, such as papers reporting studies in which the researchers produce recombinant proteins from the organism of interest rather than from cell extract derived from that organism.

Figure 4. High adoption cell-free platforms and their applications. (A) Web of the applications enabled by high adoption cell-free platforms. The connections shown are based on applications that have been published for each respective platform. Applications under “difficult to synthesize proteins” include the production of antibodies, large proteins, ice structuring proteins, and metalloproteins. Miscellaneous applications include studies of translational machinery, genetic circuits, metabolic engineering, and genetic code expansion. (B) Cumulative number of peer-reviewed publications over the last 60 years for high adoption platforms. The metric of cumulative publications by platform is used to indicate which platforms are most utilized, with platforms having over 25 papers categorized as high adoption. These data were generated by totaling papers from a PubMed Boolean search of the following: (“cell free protein synthesis” OR “in vitro transcription translation” OR “in vitro protein synthesis” OR “cell free protein expression” OR “tx tl” OR “cell-free translation”) AND “platform name.” The platform name used for each search corresponds to the name listed in the graph’s key. This information was collected on 23 December 2018, and the search results for each platform can be found in Supplemental Information. While this metric is an indicator of the level of adoption for each platform, it does suffer from false positive search results, such as papers reporting studies in which the researchers produce recombinant proteins from the organism of interest rather than from cell extract derived from that organism.

Figure 5. Low adoption cell-free platforms and their applications. (A) Web of the applications enabled by low adoption cell-free platforms. Connections shown are based on applications that have been published or that have been proposed in publications. Applications under “difficult to synthesize proteins” include high GC content proteins, antimicrobial peptides, pharmaceutical proteins, and thermophilic proteins. Miscellaneous applications include studies of translational machinery, investigation of antibiotic resistance, genetic circuits, metabolic engineering, and genetic code expansion. (B) Cumulative number of peer-reviewed publications over the last 60 years for low adoption platforms. We have used the metric of cumulative publications to indicate which platforms are less utilized and have categorized platforms with under 25 papers as low adoption platforms. These data were generated by totaling papers from a PubMed Boolean search of the following: (“cell free protein synthesis” OR “in vitro transcription translation” OR “in vitro protein synthesis” OR “cell free protein expression” OR “tx tl” OR “cell-free translation”) AND “platform name.” The platform name used for each search corresponds to the name listed in the graph’s key. While this metric is an indicator of the level of adoption for each platform, it does suffer from inconsistencies due to irrelevant search results, such as papers reporting studies in which the researchers produce proteins from the organism of interest rather than from cell extract derived from the organism. This inconsistency was significant for platforms with fewer papers, so we pursued data curation to remove irrelevant papers and add in missing papers. This information was collected on 23 December 2018, and curated search results for each platform can be found in Supplemental Information, where red indicates that the paper was removed from the search results and green indicates that the paper was added to the search results.

Figure 5. Low adoption cell-free platforms and their applications. (A) Web of the applications enabled by low adoption cell-free platforms. Connections shown are based on applications that have been published or that have been proposed in publications. Applications under “difficult to synthesize proteins” include high GC content proteins, antimicrobial peptides, pharmaceutical proteins, and thermophilic proteins. Miscellaneous applications include studies of translational machinery, investigation of antibiotic resistance, genetic circuits, metabolic engineering, and genetic code expansion. (B) Cumulative number of peer-reviewed publications over the last 60 years for low adoption platforms. We have used the metric of cumulative publications to indicate which platforms are less utilized and have categorized platforms with under 25 papers as low adoption platforms. These data were generated by totaling papers from a PubMed Boolean search of the following: (“cell free protein synthesis” OR “in vitro transcription translation” OR “in vitro protein synthesis” OR “cell free protein expression” OR “tx tl” OR “cell-free translation”) AND “platform name.” The platform name used for each search corresponds to the name listed in the graph’s key. While this metric is an indicator of the level of adoption for each platform, it does suffer from inconsistencies due to irrelevant search results, such as papers reporting studies in which the researchers produce proteins from the organism of interest rather than from cell extract derived from the organism. This inconsistency was significant for platforms with fewer papers, so we pursued data curation to remove irrelevant papers and add in missing papers. This information was collected on 23 December 2018, and curated search results for each platform can be found in Supplemental Information, where red indicates that the paper was removed from the search results and green indicates that the paper was added to the search results.

Figure 6. General workflow for preparation of cell-free extract and set up of CFPS reactions. A visualization from cell growth to the CFPS reaction is depicted above for a new user, highlighting the main steps involved.

Figure 6. General workflow for preparation of cell-free extract and set up of CFPS reactions. A visualization from cell growth to the CFPS reaction is depicted above for a new user, highlighting the main steps involved.

Table 1. Comparison of growth methods for high adoption platforms. We have assembled the major growth methodologies for each of the high adoption platforms to give users an idea of the relative differences between them. These are not the only techniques that have been used for growth for each platform, but they are generally representative of the methods.

Table 1. Comparison of growth methods for high adoption platforms. We have assembled the major growth methodologies for each of the high adoption platforms to give users an idea of the relative differences between them. These are not the only techniques that have been used for growth for each platform, but they are generally representative of the methods. Growth Platform Media/Vessel Harvest Key Citations E. coli Media: 2× YPTG (5 g NaCl, 16 g Tryptone, 10 g Yeast extract, 7 g KH 2 PO 4 , 3 g KHPO 4 , pH 7.2/750 mL solution, 18 g Glucose/250 mL solution).

Vessel: 2 L Baffled Flask.

Conditions: 37 °C, 200 RPM When OD 600 is 3, centrifuge at 5000× g for 10 min at 10 °C. Wash pellet with 30 mL S30 buffer (10 mM Tris OAc, pH 8.2, 14 mM Mg(OAc) 2 , 60 mM KOAc, 2 mM DTT), then centrifuge at 5000× g for 10 min at 10 °C. Repeat wash 3 times in total. [22] Wheat Germ Grind wheat seeds in a mill. Sieve through 710–850 mm mesh, select embryos via solvent flotation method using a solvent containing 240:600 v / v cyclohexane and carbon tetrachloride. Dry in fume hood overnight. [194] Yeast Media: 2% w / v Peptone, 1% w / v Yeast extract, 2% w / v Glucose

Vessel: 2.5 L baffled flask

Conditions: 30 °C, 250 RPM When OD 600 of 10–12 is reached, centrifuge culture for 10 min at 3000× g . Wash pellet with Buffer A (20 mM HEPES, pH 7.4, 100 mM KOAc, 2 mM Mg(OAc) 2 ). Centrifuge for 10 min at 3000× g and 4 °C. Repeat washing 3 times. [19] Rabbit Reticulocyte Make rabbits anemic over 3 days by injections of APH. Bleed rabbits on day 8. Filter blood through cheesecloth and keep on ice, then centrifuge at 2000 RPM for 10 min. [98] Insect Media: Animal component free insect cell medium.

Vessel: Fermentor.

Conditions: 27 °C When cell density reaches 4 × 106 cell/mL, centrifuge culture at 200× g for 10 min. Wash once with buffer (40 mM HEPES KOH, pH 7.5, 100 mM KOAc, 4 mM DTT). [129] HeLa Media: Minimal essential medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 1 U/mL penicillin, 0.1 mg/mL streptomycin.

Vessel: Spinner flask with cell culture controller

Conditions: 37 °C, pH 7.2, 67 ppm oxygen, 50 RPM Harvest when cell density reaches 0.7–0.8 × 106 cells/mL. Wash 3 times with buffer (35 mM HEPES KOH, pH 7.5, 140 mM NaCl, 11 mM glucose). [13] Chinese Hamster Ovary Media: Power Chinese hamster ovary-2 chemically defined serum-free media

Vessel: Fermentor

Conditions: 37 °C Harvest at 2 × 106 cells/mL cell density by centrifuging at 200× g for 10 min. Wash cells once with buffer (40 mM HEPES KOH, pH 7.5, 100 mM NaOAc, 4 mM DTT). [17]

Table 2. Comparison of extract preparation methods for high adoption platforms. We have assembled the major extract preparation methodology for each of the high adoption platforms to give users an idea of the relative differences between them. These are not the only techniques that have been used for extract preparation for each platform, but they are generally representative of the methods.

Table 2. Comparison of extract preparation methods for high adoption platforms. We have assembled the major extract preparation methodology for each of the high adoption platforms to give users an idea of the relative differences between them. These are not the only techniques that have been used for extract preparation for each platform, but they are generally representative of the methods. Extract Prep Platform Pre-Lysis Lysis Post-Lysis Processing Growth and Prep Time Key Citations E. coli Resuspend in 1 mL/1 g pellet of S30 buffer by vortexing. Sonicate on ice for 3 cycles of 45 s on, 59 s off at 50% amplitude. Deliver 800–900 J total for 1.4 mL of resuspended pellet. Supplement with a final concentration of 3 mM DTT. Centrifuge lysate at 18,000× g and 4 °C for 10 min. Transfer supernatant while avoiding pellet. Perform runoff reaction on supernatant at 37 °C and 250 RPM for 60 min. Centrifuge at 10,000× g and 4 °C for 10 min. Flash freeze supernatant and store at −80 °C. 1–2 days [22] Wheat Germ Wash 3 times with water under vigorous stirring to remove endosperm. Sonicate for 3 min in 0.5% Nonidet P-40. Wash with sterile water. Grind washed embryos into fine powder in liquid nitrogen and resuspend 5 g in 5mL of 2× Buffer A (40 mM HEPES, pH 7.6, 100 mM KOAc, 5 mM Mg(OAc) 2 , 2 mM CaCl, 4 mM DTT, 0.3 mM of each of the 20 amino acids). Centrifuge at 30,000× g for 30 min. Filter supernatant through G-25 column equilibrated with Buffer A. Centrifuge column product at 30,000× g for 10 min. Adjust to 200 A 260 /mL with Buffer A. Store in liquid nitrogen. 4–5 days [64,194] Yeast Resuspend cells in 1 mL lysis buffer (20 mM HEPES KOH, pH 7.4, 100 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT, 0.5 mM PMSF) per 1 g cell pellet. Pass through homogenizer once at 30,000 psig. Centrifuge homogenate at 30,000× g for 30 min at 4 °C. Then repeat centrifugation with supernatant in a spherical bottom bottle. Desalt supernatant in dialysis tubing with 4 exchanges of 50× volume lysis buffer for 30 min each at 4 °C. Centrifuge at 60,000× g for 20 min at 4 °C. Flash freeze and store at −80 °C. 1–2 days [19,195] Rabbit Reticulocyte Resuspend cells in buffered saline with 5 mM glucose, then centrifuge at 2000 RPM for 10 min. Repeat wash 3 times. Resuspend cells in 1.5 times the packed cell volume of ice-cold water, mix thoroughly. Spin lysate at 15,000× g for 20 min at 2 °C. Pour supernatant though 53 μm nylon. Treat with micrococcal nuclease by adding 0.2 mL of 1 mM hemin, 0.1 mL of 5 mg/mL creatine kinase, 0.1 mL of 0.1 M CaCl 2 , 0.1 mL of micrococcal nuclease. Incubate at 20 °C for 15 min, then add 0.1 mL of 0.2 M EGTA and 60 μL of 10 mg/mL tRNA. Store in liquid nitrogen or at −70 °C. ~8 days to treat rabbits, 1 day for extract preparation [98] Insect Resuspend cells in buffer to final density of 2 × 108 cells/mL. Mechanically lyse cells by rapidly freezing in liquid nitrogen, then thawing in water bath at 4 °C or use a disruption chamber with 20 kg/cm2 nitrogen gas for 30 min. Centrifuge lysate at 10,000× g for 10 min. Apply supernatant to G-25 gel filtration column. Pool fractions with highest A 260 , flash freeze in liquid nitrogen and store at −80 °C. 1–2 days [18,129,195,196] HeLa Resuspend in extraction buffer (20 mM HEPES KOH, pH 7.5, 135 mM KOAc, 30 mM KCl, 1.655 mM Mg(OAc) 2 ) to ~2.3 × 108 cells/mL. Disrupt cells via 1 MPa nitrogen pressure for 30 min in a cell disruption chamber. Centrifuge homogenate at 10,000× g for 5 min at 4 °C. Pass supernatant through a PD-10 desalting column equilibrated with extraction buffer. Treat 100 μL of extract with 1 μL of 7500 U/mL nuclease S7 and 1 μL of 100 mM CaCl 2 for 5 min at 23 °C, then add 8 μL of 30 mM EGTA. Flash freeze eluted extract in liquid nitrogen and store at −80 °C. 1–2 days [13,195] Chinese Hamster Ovary Resuspend cells in buffer to density of 5 × 108 cells/mL. Disrupt cells by syringing the pellet through a 20-gauge needle. Centrifuge lysate at 10,000× g for 10 min. Apply supernatant to G-25 gel filtration column. Pool fractions with an A 260 above 100. Treat pooled fractions with 10 U/mL S7 nuclease and 1 mM CaCl 2 , incubate at room temperature for 2 min, then add 6.7 mM EGTA. Flash freeze in liquid nitrogen and store at −80 °C. 1–2 days [17]

Table 3. Comparison of cell-free protein synthesis reaction setup for high adoption platforms. This table is intended to help users understand major differences between setups for various high adoption platforms, namely whether reactions are generally coupled or uncoupled, what energy systems are typical, and what temperatures the reactions are run at. These are not the only setups that have been used for successful cell-free protein expression in each platform, but they are generally representative of the reagents, concentrations, and temperatures used for each platform.