OnePot PURE Cell-Free System

Summary

We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

Abstract

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

Introduction

Cell-free transcription-translation (TX-TL) systems constitute a promising platform for investigating and engineering biological systems. They provide simplified and tunable reaction conditions, as they no longer rely on life-sustaining processes, including growth, homeostasis, or regulatory mechanisms¹. Thus, it is anticipated that cell-free systems will contribute to the investigation of biomolecular systems, offer a framework to test rational biodesign strategies², and provide a chassis for a future synthetic cell^3,4. The fully recombinant PURE system offers an especially appealing chassis due to its defined and minimal composition, as well as its adjustability and tuneability⁵.

Since the first functional, fully recombinant PURE system was established in 2001⁵, efforts have been made to expand the system limits and optimize the system's composition to improve the system yields^6,7,8, allow for transcriptional regulation⁹, membrane^10,11 and secretory protein synthesis¹², and to facilitate protein folding^13,14. Nowadays, there are three commercially available systems: PUREfrex (GeneFrontier), PURExpress (NEB), and Magic PURE (Creative Biolabs). However, those systems are costly, their exact composition is proprietary and thus unknown, and adaptability is limited.

PURE systems prepared in-house proved to be the most cost-effective and tunable option^15,16. However, the required 37 purification steps for protein and ribosome fractions are time-consuming and tedious. Several attempts have been made to improve the efficiency of the PURE system preparation^17,18,19. We recently demonstrated that it is possible to coculture and co-purify all required non-ribosomal proteins present in the PURE system. This OnePot method has proved to be cost-effective and time-efficient, cutting down preparation time from several weeks to 3 working days. The approach generates a PURE system with a protein production capacity comparable to the commercially available PURExpress system²⁰. Contrary to the previous approaches to simplify the PURE preparation^17,18,19, in the OnePot approach all proteins are still expressed in separate strains. This enables the user to tune the composition of the OnePot PURE system by merely omitting or adding specific strains or adjusting the inoculation volumes, thus generating dropout PURE systems or altering the final protein ratios, respectively.

The protocol presented here provides a detailed method for creating the OnePot PURE system as described previously²⁰, although β-mercaptoethanol was replaced with tris(2-carboxyethyl)phosphine (TCEP). Moreover, two methods for ribosome purification are described: traditional tag-free ribosome purification using hydrophobic interaction and sucrose cushion, adapted from Shimizu et al.¹⁵, and Ni-NTA ribosome purification based on Wang et al.¹⁸ and Ederth et al.²¹ but significantly modified. The latter method further facilitates the preparation of the PURE system and makes it accessible to more laboratories, as only standard laboratory equipment is required.

The experimental protocol summarizes the preparation of a versatile PURE cell-free TX-TL system to provide a simple, tunable, cost-effective cell-free platform, which can be prepared using standard laboratory equipment within a week. Besides introducing the standard PURE composition, we indicate how and where it can be adjusted, with a primary focus on critical steps in the protocol to ensure the system's functionality.

Protocol

NOTE: This protocol describes the preparation of cell-free TX-TL system from recombinant components. For convenience, the work is separated into five parts. The first part describes preparation steps, which should be done before starting the protocol. The second part describes the preparation of the OnePot protein solution. The third part describes ribosome purifications, the fourth part details the preparation of the energy solution, and the last part provides a manual for setting up a PURE reaction. For convenience, the protocols are divided into days and summarized in daily schedules in Table 1. Following the schedule, the whole system can be prepared in 1 week by one person.

1. Preliminary work

1. Prepare the bacterial culture media and media supplements as described in Supplementary Table 1. Prepare and sterilize the materials required, including pipette tips, 96 deep-well plates.
2. Strain preparation
   1. Transform the expression strains indicated in Table 2 with the corresponding expression vectors using the heat shock method.
      1. Add purified plasmid to the chemically competent bacteria and incubate on ice for 20-30 min.
      2. Place the mixture at 42 °C for 30 s (heat shock) and then place it back on ice for 2 min.
      3. Pipette 20 µL of the bacteria directly onto agar plates containing ampicillin (AMP) and incubate at 37 °C overnight. Store the plates at 4 °C for up to 1 week.
      4. Inoculate 3 mL of LB media containing AMP with a single colony of bacteria from the agar plates. Incubate at 37 °C while shaking at 260 rpm overnight.
      5. Mix 250 µL of the culture with 250 µL of 50% (v/v) glycerol and store at -80 °C.
         NOTE: For faster preparation in the future, store the strains in a 96-well plate as glycerol stocks.
   2. Confirm all vector transformations by colony PCR and sequencing. Sequence the gene, promoter region, and ribosome binding site.
3. Expression test
   1. Inoculate 300 µL of LB media containing AMP with around 1 µL of the prepared glycerol stocks in a 1.3 mL deep-well plate. Seal the plate with a breathable membrane and then incubate at 37 °C while shaking at 260 rpm overnight.
      NOTE: All expressions are done separately at this point.
   2. Inoculate 300 µL of fresh LB media containing AMP with 1 µL of the overnight cultures. Incubate at 37 °C while shaking at 260 rpm overnight. After 2 h, induce the cells with 100 µM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) and grow for an additional 3 h.
   3. Mix 10 µL of the culture with 10 µL of 2x Laemmli buffer and heat to 95 °C for 10 min. Spin the samples for 1 min using a table centrifuge and load 10 µL of the supernatant on a PAGE gel. Run the gel in Tris/Glycine/SDS buffer at 200 V for 30 min. Rinse it well with deionized water. Cover the gel with a Coomassie protein stain and incubate for 1 h. Destain the gel in water if necessary (representative results for the expression test in Figure 1).
      NOTE: Use gradient (4%-15% or 4%-20%) PAGE gels to achieve a good separation.
4. IMAC Sepharose resin restoration and cleaning
   1. Column preparation.
      1. Mix the Sepharose resin well by vortexing.
      2. Pipette the required amount of resin into an empty gravity flow column.
         NOTE: The amount of resin required varies between His-ribosome purification and protein purification and is specified in the respective sections.
      3. Wash the resin with 30 mL of deionized water.
      4. Proceed with column re-charge as specified in section 1.4.4.
         NOTE: Always let all the liquid pass through the column before continuing with the next step. However, make sure that the column never runs dry. Whenever running any liquid through the column, ensure to stop the flow or continue to the next step as soon as the liquid reaches the resin.
   2. Restoration.
      1. Wash the column with 30 mL of deionized water.
      2. Apply 10 mL of a 0.2 M EDTA and 0.5 M NaCl solution.
      3. Add 30 mL of a 0.5 M NaCl solution.
      4. Wash the column with 50 mL of deionized water.
      5. Store in 20% (v/v) ethanol at 4 °C or continue with the next step.
   3. Cleaning.
      CAUTION: Wear protective equipment.
      1. Wash the column with 30 mL of 0.5 M NaOH.
      2. Wash the column with 30 mL of deionized water.
      3. Wash the column with 30 mL of 0.1 M acetic acid.
      4. Wash the column with 30 mL of deionized water.
      5. Wash the column with 30 mL of 70% (v/v) ethanol.
      6. Wash the column with 50 mL of deionized water.
      7. Store in 20% (v/v) ethanol at 4 °C or continue with the next step.
   4. Re-charging.
      1. Add 10 mL of 0.1 M nickel sulfate solution to the column.
         CAUTION: Nickel sulfate is toxic. Nickel sulfate waste needs to be discarded with the precautions indicated by the supplier.
      2. Wash the column with 50 mL of deionized water.
      3. Store in 20% ((v/v)) ethanol at 4 °C or continue with the column equilibration.
         ​NOTE: If the column is stored in ethanol between steps, make sure to remove all traces of ethanol by washing the column with water.

2. OnePot protein solution expression and purification

NOTE: The protocol consists of three parts divided into days (Figure 2). An ideal preparation procedure produces 1.5 mL of 13.5 mg/mL OnePot protein solution, which corresponds to more than one thousand 10 µL PURE reactions. However, the amount and the ideal concentration of the solution will vary from batch to batch. Experienced users can perform multiple OnePot PURE preparations at a time.

Day 1:

1. Prepare bacterial culture media and media supplements as described in Supplementary Table 1.
2. Prepare and sterilize the required materials, including pipette tips, two 96 deep-well plates, and one 1 L baffled Erlenmeyer flask.
3. Prepare buffers and supplements as described in Supplementary Table 2. Filter sterilize all buffers using bottle top filters (0.45 µm) and store them at 4 °C. Supplement all the buffers with 1 mM TCEP right before use, unless indicated otherwise.
4. Use 2 mL of sepharose resin for the OnePot protein purification. Prepare the column as described in section 1.4.
5. To prepare the starter cultures, combine 20 mL of LB media with 20 µL of AMP. In a sterile 96, 1.3 mL deep-well plate, add 300 µL of the media into 35 wells. Inoculate each of them with its respective strain, except elongation factor thermo unstable (EF-Tu), and seal the plate with a breathable membrane.
   NOTE: Inoculate the plate using a 96-well replicator (see Table of Materials). The well volume of the deep-well plate and the volume of the starter culture are essential. Larger media volumes or smaller well volumes will lead to a different bacterial density due to aeration inconsistencies.
6. For the EF-Tu culture, inoculate 3 mL of LB media in a 14 mL culture tube with a snap cap. A single 3 mL of culture for EF-Tu is sufficient for one OnePot expression culture.
7. Incubate at 37 °C while shaking at 260 rpm overnight.

Day 2:
NOTE: Perform all the steps at room temperature unless indicated otherwise.

8. Transfer 500 mL of LB media and 500 µL of AMP into the sterile baffled flask.
9. Inoculate the OnePot PURE culture with 1675 µL of the EF-Tu culture and 55 µL of each of the cultures from the deep-well plate (Table 2).
   NOTE: During this step, the overall protein composition can be adjusted by tuning the inoculation ratios. Make sure that the overall inoculation volume remains constant at 3.6 mL.
   OPTIONAL: To confirm that all strains have grown overnight, measure the optical density of the overnight cultures at 600 nM (OD₆₀₀) in a 96-well plate using a plate-reader. Use a dilution of 10x for the optical density measurement.
10. Incubate the culture for 2  h at 37 °C with a shaking of 260 rpm, or until the OD₆₀₀ of the culture reaches 0.2-0.3.
11. Induce the culture with 500 µL of 0.1 mM IPTG and grow for an additional 3 h.
12. Harvest the cells by centrifugation at 4 °C and 3220 x g for 10 min and store the cell pellet at -80 °C until further use.
    NOTE: To optimize the timing, prepare the energy solution described in section 4 during the incubation times on day 2 (Table 1).

Day 3:

13. Measure the amounts of buffers needed for the purification described in the steps below and add TCEP to all of them as indicated in Supplementary Table 2. Store the remaining buffers without TCEP at 4 °C for future purifications.
14. Equilibrate the charged column (section 2.4) with 30 mL of buffer A. After 25 mL of buffer A have passed through, close the column from the bottom. In parallel, continue with steps 2.15-2.17.
15. Thaw the cells and use a serological pipette to resuspend the cell pellet in 7.5 mL of buffer A.
16. Lyse the cells using a 130-watt probe sonicator (see Table of Materials, probe tip diameter: 6 mm) with the following parameters: 4 x 20 s pulse on, 20 s pulse off, 70% amplitude. If sonication is successful, the solution will turn darker (Figure 2).
    NOTE: Make sure to keep the cells on ice during sonication. Place the probe deep enough into the solution without touching the tube. If a large amount of foam is generated, the energy transfer will be damped. In that case, let the foam settle, lower the probe deeper into the solution, and extend the sonication time.
17. Remove the cell debris by centrifugation at 21130 x g for 20 min at 4 °C immediately after sonication. Keep the lysate on ice.
18. Add the supernatant to the equilibrated column. Close the column from the top and make sure there is no leakage. Incubate the column for 3  h at 4 °C under rotation using a tube rotator.
19. Elute unbound components from the column and wash with 25 mL of buffer A.
20. Wash the column with 25 mL of 25 mM imidazole buffer (23.95 mL of buffer A and 1.25 mL of buffer B).
21. Elute the proteins with 5 mL of 450 mM imidazole buffer (0.5 mL of buffer A and 4.5 mL of buffer B). Keep the eluted proteins on ice at all times.
22. Dilute the eluate with 25 mL of HT buffer, keep the mixture on ice. Add 15 mL to a 15 mL centrifugal filter and concentrate to a volume of 1.5 mL. Add the remaining 15 mL to the filter with the concentrated solution and concentrate to 1.5 mL once more.
23. Add 10 mL of HT buffer to the concentrated sample and concentrate to 1 mL. Add an equal amount of stock buffer B and store at -80 °C until further use.
    NOTE: One round of exchange/concentration takes about 60 min spinning at 3220 x g at 4 °C.
24. During the buffer exchange, restore the column as specified in section 1.4.

Day 4:

25. Measure the protein concentration using the Bradford assay as described by the supplier. Concentrate the sample with a 0.5 mL of 3 kDa cutoff centrifugal filter to 20 mg/mL.
    NOTE: Dilute the protein solution 25-fold or 50-fold before the concentration measurements to avoid oversaturating the Bradford assay.
26. To establish the ideal protein concentration, perform an expression test at this stage (section 5.2) with different concentrations of the protein solution. To perform the titration, keep the total volume of the solution constant and pipette the OnePot protein solution, including stock buffer B, at five different ratios (Supplementary Table 7).
27. Verify the OnePot PURE protein composition using SDS-PAGE (Figure 3A). Dilute 2.5 µL of the sample with 7.5 µL of water, mix with 10 µL of 2x Laemmli buffer and then load 5 µL and 2.5 µL of the samples to the gel. Run the SDS-PAGE as specified in section 1.3.3.
28. Aliquot the protein solution into 50 µL aliquots after verifying the expression and adjusting the concentration. Store the OnePot PURE protein solution at -80 °C until further use.
    NOTE: If a protein component is suspected not to be present, or is present in a lower-than-expected concentration in the OnePot PURE, perform the following steps.
29. Check whether the overnight culture of the respective strain has grown at a comparable rate to the other cultures by performing optical density measurements (OD₆₀₀) of all cultures.
30. Perform an additional expression test of the specific strain to verify the expression of the suspect protein.

3. Ribosome solution

NOTE: Two different ribosome purification strategies are introduced, one for hexahistidine-tagged and one for non-tagged ribosomes. The major advantage of the purification method using His-purification on a standard affinity Ni-NTA gravity flow column is that the purification is easy, fast, and does not require additional laboratory equipment, such as a FPLC system and an ultracentrifuge. However, the protein production capacity in OnePot PURE reactions is around one-third compared to tag-free ribosomes. Therefore, choose the method for ribosome production based on whether a high yield is important for the given application.

1. His-tagged ribosome purification
   NOTE: This protocol utilizes the E. coli RB1 strain, a gift from Professor Wang (Columbia University, USA)¹⁸. This strain has a genomic insertion of a hexahistidine tag on the C terminus of 50S ribosomal protein (L7/L12), allowing for purification using a Ni-NTA gravity-flow column. The usual yield is around 0.5 mL of 3.45 µM ribosomes, which is sufficient for more than five hundred 10 µL PURE reactions.

Day 1:

1. Prepare bacterial culture media and media supplements as described in Supplementary Table 1.
2. Prepare and sterilize the required materials, including pipette tips, one 5 L Erlenmeyer flask, and one 100 mL Erlenmeyer flask.
3. Prepare buffers and supplements as described in Supplementary Table 2. Filter sterilize all the buffers using bottle top filters (0.45 µm) and store them at 4 °C.

Day 2:

4. Pipette 5 mL of resin to a column and prepare the column as specified in section 1.4.
   NOTE: Due to the higher volume of the resin, the restoration and purification take significantly longer. Use a different column for ribosome purification to avoid cross-contamination and thoroughly clean it before the purification.
5. Prepare an overnight culture of E. coli RB1 strain by inoculating 35 mL of LB media in a 100 mL Erlenmeyer flask. Incubate at 37 °C while shaking at 260 rpm.

Day 3:
NOTE: Perform all the steps at room temperature unless indicated otherwise.

6. Add 2 L of LB media into a 5 L sterile flask, inoculate with 12 mL of the overnight culture, and then incubate for 3-4 h at 37 °C while shaking at 260 rpm.
   NOTE: Alternatively, perform bacterial culturing in 4 x 500 mL of cultures in 1 L baffled flasks.
7. Pellet the cells by centrifugation for 10 min at 3220 x g and 4 °C. Store at -80 °C until further use.

Day 4:

8. Equilibrate the column prepared in step 3.1.4. with 30 mL of lysis buffer.
9. Resuspend the pellet in 20 mL of lysis buffer using a serological pipette.
10. Lyse the cells with a 130-watt probe sonicator (see Table of Materials, probe tip diameter: 6 mm) on ice with the following parameters: 11 x 20 s pulse on; 20 s pulse off, 70% amplitude (see step 2.16 for procedure details).
11. Immediately after sonication, remove the cell debris by centrifugation for 20 min at 21130 x g at 4 °C. Keep the lysate on ice.
12. Load the supernatant to the columns and let it pass through.
13. Wash the column with the following mixtures of lysis and elution buffers.
    1. Wash 0: use 30 mL of lysis buffer.
    2. Wash 1: use 30 mL of 5 mM imidazole (29 mL of lysis buffer, 1 mL of elution buffer).
    3. Wash 2: use 60 mL of 25 mM imidazole (50 mL of lysis buffer, 10 mL of elution buffer).
    4. Wash 3: use 30 mL of 40 mM imidazole (22 mL of lysis buffer, 8 mL of elution buffer).
    5. Wash 4: use 30 mL of 60 mM imidazole (18 mL of lysis buffer, 12 mL of elution buffer).
14. Elute the ribosomes with 7.5 mL of the elution buffer. Keep the eluted proteins on ice at all times.
15. Add 22 µL of pure β-mercaptoethanol to 45 mL of ribosome buffer.
    CAUTION: β-mercaptoethanol is toxic. Take safety precautions and work in a fume hood.
16. Add the eluate to a 15 mL centrifugal filter and concentrate to 1 mL.
17. Add 15 mL of ribosome buffer to the concentrated sample and concentrate again to 1 mL.
    NOTE: Repeat the previous step twice.
18. Store at -80 °C until further use.
    ​NOTE: One round of exchange/concentration takes about 60 min of centrifugation at 3220 x g at 4 °C.
19. During the buffer exchange, restore the column as specified in section 1.4.

Day 5:

21. Determine the ribosome concentration by measuring the absorbance at 260 nM of a sample diluted 1:100 in ribosome buffer. An absorbance value of 10 of the diluted solution corresponds to 23 µM of undiluted solution as previously described¹⁶.
22. Implement a final stock concentration of 3.45 µM. To adjust the concentration, dilute the ribosomes with ribosome buffer or concentrate them further by centrifugation at 14000 x g in a 3 kDa 0.5 mL centrifugal filter at 4 °C.
    NOTE: To achieve optimal system expression, perform a ribosome concentration titration (section 5.2, Supplementary Table 7).
23. Verify the ribosome composition using SDS-PAGE (Figure 3A) as specified in section 1.3.3. Dilute 2.5 µL of the sample with 7.5 µL of water, mix with 10 µL of 2x Laemmli buffer, and then load 5 µL and 2.5 µL of the samples onto the gel.

2. Tag-free ribosome purification
   NOTE: Tag-free ribosome purification is performed using a FPLC system (Table of Materials) and is based on hydrophobic interaction chromatography using 2 x 5 mL Butyl columns (Table of Materials). Although ribosomes may be purified from any strain, using the E. coli A19 (E. coli Genetic Resources at Yale CGSC) strain is advantageous due to its RNase I deletion²². Perform the purification at 4 °C in either a cold room or a cooling cabinet. The usual yield is around 0.5 mL of 10 µM ribosomes, which corresponds to more than five hundred 10 µL PURE reactions.

Day 1:

1. Prepare bacterial culture media and media supplements as described in Supplementary Table 1.
2. Prepare and sterilize the required materials, including pipette tips, 5 L Erlenmeyer flask, and 100 mL Erlenmeyer flask.
3. Prepare buffers and supplements as described in Supplementary Table 2. Filter sterilize all the buffers using bottle top filters (0.45 µm) and store them at 4 °C.

Day 2:

4. To prepare an overnight culture of the E. coli A19 strain, inoculate 35 mL of LB media in a 100 mL Erlenmeyer flask. Incubate at 37 °C while shaking at 260 rpm.

Day 3:

5. Transfer 2 L of LB media into the 5 L sterile baffled flask, inoculate with 30 mL of the overnight culture, and then incubate for 3-4  h at 37 °C while shaking at 200 rpm.
6. Pellet the cells by centrifugation at 4000 x g for 15 min at 4 °C. Resuspend the pellet in 25 mL of suspension buffer and store at -80 °C until further use.

Day 4:

7. Perform steps 3.2.8-3.2.12 in parallel with steps 3.2.13-3.2.19.
8. Thaw and lyse the cells using a 130-watt probe sonicator (see Table of Materials and probe tip diameter: 6 mm) on ice with the following parameters: 12 x 20 s pulse on; 20 s pulse off, 70% amplitude (see step 2.16 procedure details).
9. Immediately remove the cell debris by centrifugation at 20000 x g for 20 min at 4 °C.
10. Aspirate the supernatant and measure the volume. Add an equal volume of suspension buffer (high salt) to adjust the final concentration of ammonium sulfate to 1.5 M and mix well.
11. Remove the precipitate by centrifugation at 20000 x g for 20 min at 4 °C.
12. Filter the supernatant using a 0.45 µm polyethersulfone membrane syringe filter before FPLC purification and collect the filtrate in a 100 mL glass bottle. Keep the supernatant at 4 °C at all times.
13. Set up the FPLC system for hydrophobic-interaction chromatography purification using a double Butyl column (2 x 5 mL) as follows. For this setup, one column volume (CV) refers to a volume of 10 mL.
14. Three inlets will be needed: two as buffer lines and one as the sample line. Due to the default settings of the purifier, it is convenient to choose lines A1 and B1 for buffer C and buffer D, respectively, and line A2 as the sample line. Apply a default flow rate of 4 mL/min, except for pump washes (10 mL/min) or unless indicated otherwise.
    NOTE: As TCEP is a costly reagent, add the corresponding amount to buffers C and D only after the equilibration step.
15. Perform a system pump wash in 20% ((v/v)) ethanol to clean the system and remove potential contamination from previous purifications. Manually set a flow rate of 0.2 mL/min and mount the column. Stop the flow.
16. Execute a system pump wash with water. Wash the column with 3 CV of water.
17. Equilibration: place inlets A1 and A2 in buffer C and inlet B1 in buffer D without TCEP. Execute a pump wash and equilibrate the column with 4 CV of buffer C.
18. Add TCEP to buffers C and D.
19. Prepare 15 mL tubes or clear round fraction collector tubes to the fraction collector to collect 4-5 mL elution fractions.
20. Loading: Place the inlet A2 into the bottle with the filtered sample. Load approximately 90% of the sample volume onto the column. Dilute the sample with 20 mL of TCEP-containing buffer C, and load 10 mL of the sample onto the column. Repeat the dilution step at least twice and load as much sample onto the column as possible. It is critical to ensure that no air is sucked into the machine.
21. Washing step 1: wash with 3 CV of buffer C to remove the unbound components.
22. Washing step 2: wash with 5 CV of 80% buffer C and 20% buffer D.
23. Elution: elute the product by applying 50% of buffer C and 50% of buffer D, with a total elution volume of 5 CV. Collect this fraction in the collector tubes.
24. Washing step 3: Elute all strongly interacting contaminants using 100% buffer D with a total volume of 5 CV.
25. Analyze the absorption spectrum of the sample fraction at 260 or 280 nM (Figure 4). The first peak shows the non-absorbed proteins eluted during loading and the first washing step; the second peak shows contaminants that have been eluted during the second washing step. The third peak monitors the final product, and the last peak shows the strongly interacting contaminants. Pool all sample fractions corresponding to the third peak for further processing. Keep the eluted proteins on ice at all times.
26. Gently overlay the recovered fraction onto 15 mL of the cushion buffer in four polycarbonate ultracentrifugation tubes. Add a maximum of 15 mL of the sample to 15 mL of the cushion buffer. Make sure to balance the weight of the tube well. Pellet the ribosomes by ultracentrifugation at 100000 x g at 4 °C for 16 h.
    NOTE: Ensure that no cracks are present in the ultracentrifugation tubes.
27. Clean and reset the column as follows. A flow rate of 5 mL/min works well. Place all the inlets into the water and execute a pump wash. Wash the column with 2 CV of water.
    1. Place the inlet into a 0.5 M NaOH solution, perform a pump wash, and subsequently wash the column with 3 CV of NaOH.
    2. Place the inlet into water, perform a pump wash, and then wash the column in 2 CV of water.
    3. Place the inlet to a 0.1 M acetic acid solution, perform a pump wash, and subsequently wash the column with 3 CV of acetic acid solution.
    4. Pump wash and wash the column with 2 CV of water.
    5. Place all inlets into 20% ((v/v)) ethanol, execute a pump wash step, and store the column in 20% ((v/v)) ethanol by washing it with 3 CV of a 20% ((v/v)) ethanol solution.
       NOTE: Ensure that the system never runs dry or sucks in air. Never apply buffer directly to ethanol, or ethanol to buffer. Always add a water washing step in between, as otherwise there is a risk of precipitates clogging the column. Make sure to add enough sample collection tubes.

Day 5:

28. Discard the supernatant and carefully, without disturbing the translucent pellet, wash each pellet with 0.5 mL of ice-cold ribosome buffer. Repeat this step twice.
29. Resuspend each of the clear pellets in 100 µL of ribosome buffer on ice using a magnetic stir bar (3 mM diameter, 10 mM length) on a magnetic stirrer using the lowest possible speed. Collect the resuspended ribosomes and wash the tubes with an additional 50 µL of ribosome buffer.
    NOTE: The translucent pellet is difficult to see. Therefore, carefully wash the pellet from the sides of the tube.
30. Determine the ribosome concentration by measuring the absorbance at 260 nM of the sample diluted at a ratio of 1:100 in ribosome buffer. An absorbance of 10 of the diluted solution corresponds to 23 µM of undiluted solution as previously described¹⁶.
31. Implement a final stock concentration of 10 µM. To adjust the concentration, dilute the ribosomes with ribosome buffer or concentrate them further by centrifugation at 14000 x g in a 3 kDa centrifugal filter at 4 °C.
    NOTE: To achieve optimal system expression, perform ribosome titration (section 5.2, Supplementary Table 7).
32. Verify the ribosome composition with SDS-PAGE (Figure 3A) as specified in section 1.3.3. Dilute 2.5 µL of the sample with 7.5 µL of water, mix with 10 µL of 2x Laemmli buffer, and then load 5 µL and 2.5 µL of the samples to the gel.

4. Energy solution

NOTE: The composition for the 2.5x energy solution introduced here is an example of a solution that worked well for a standard TX-TL reaction. To optimize the timing, prepare the energy solution during day 2. The preparation of the amino acid solution is explained in detail, followed by the final preparation procedure.

1. Amino acid solution
   NOTE: Prepare the amino acid solution in bulk. Preparing the amount of amino acid stock solutions required for a final volume of at least 2000 µL will reduce the weighing error for the otherwise very small amounts. The overall concentration of the amino acid solution is limited by the solubility of the amino acids and the respective stock solution concentrations. For the standard PURE system, prepare a solution with a final concentration of 3.25 mM. Use the amino acid solution calculation table (Supplementary Table 3) as a template. Use cysteine in the salt form to ensure sufficient solubility. Avoid using KOH-based amino acid preparation methods. It is possible to directly weigh the exact amounts of amino acids into the final amino acid solution without preparing stock solution for all the amino acids. However, this is more challenging and less precise.
   1. Prepare stock solutions for each amino acid as described in Supplementary Table 3, except for Tyrosine.
      NOTE: Due to the different solubilities of the amino acids in water, the respective suggested concentrations of the stock solution differ.
   2. Minimal mass [mg] provides the approximate minimum mass required to obtain a sufficient amount of stock solution for the target overall volume, as a reference.
      NOTE: The minimal mass is calculated with a surplus of 10%.
   3. For an easier preparation of the solutions, do not weigh the exact amount of amino acid, but instead, for the mass at hand, adjust the amount of water to achieve the desired concentration. Calculate the amount of deionized water (Water to add [µL]) needed, based on the actual mass filled in (light yellow cells) and the desired concentration using the spreadsheet in Supplementary Table 3.
   4. Solubilize the amino acid stock solutions by vortexing until all precipitate has dissolved. The individual amino acid stock solutions can be stored at -20 °C for several weeks.
      NOTE: Some amino acids are difficult to dissolve in water; the process may take some time.
   5. Weigh the exact amount of tyrosine required to obtain a final concentration of 3.25 mM directly into the tube for the amino acid solution.
      NOTE: Tyrosine is very difficult to dissolve in water. Add it directly instead of preparing a stock solution.
   6. Add the corresponding amounts of amino acid stock solutions and water as indicated in the Final volume to add [µL] column (light blue cells) and vortex the solution well. Store the completed amino acid solution at -80 °C until further use.
2. Preparation of the energy solution
   NOTE: In total, the 2.5x energy solution contains 0.75 mM of each amino acid, 29.5 mM of magnesium acetate, 250 mM of potassium glutamate, 5 mM of ATP and GTP each, 2.5 mM of CTP, UTP, and TCEP, respectively, 8.75 mg/mL of tRNA from E. coli MRE 600, 50 mM of creatine phosphate, 0.05 mM of folinic acid, 5 mM of spermidine, and 125 mM of HEPES. First-time users prepare the energy solution in small batches of 200 µL. Store the individual solutions prepared according to Supplementary Table 4 at -20 °C or -80 °C for later use.
   1. Thaw all aqueous solutions mentioned in the Supplementary Table 5 on ice.
   2. Meanwhile, prepare the stock solutions for the remaining components listed in Supplementary Table 4. Keep all the solutions on ice after preparation.
      NOTE: Add 500 µL of RNase and DNase-free water directly to the vial to dissolve the lyophilized tRNAs. Mix well by gentle vortexing; limit pipetting to avoid introducing RNases.
   3. Add the calculated volumes (Supplementary Table 5) of stock solutions and water and mix well using a vortex. Keep the solution on ice at all times.
   4. Measure the pH of the solution by pipetting 1 µL onto a pH strip, to ensure that the pH of the solution is neutral.
   5. Aliquot the energy solution at 50-100 µL per tube on ice and store at -80 °C until further use. While aliquoting, vortex the main stock frequently to prevent the components from precipitating.
      ​NOTE: Optionally, conduct an activity assay of the newly made energy solution against commercial energy solutions, e.g. Solution A in PURExpress. If a significantly lower performance of the system with the energy solution is observed, optimizing the ion concentrations, especially magnesium ions, by titration (5-20 mM) may be advantageous.

5. OnePot PURE reaction

1. DNA template
   NOTE: Proteins encoded downstream of the T7 promoter can be expressed in PURE from either linear or circular DNA. By generating a linear DNA template using extension PCR, tedious cloning steps can be omitted. The linear templates for this study were generated by PCR as described below, using a high-fidelity DNA polymerase (Table of Materials). Primer sequences, melting temperatures, and the thermocycler settings used in this study are specified in Supplementary Table 6. The preparation of the DNA template is not included in the daily schedule.
   1. Set up a PCR reaction as recommended by the polymerase supplier.
      NOTE: Optimized parameters for a high-fidelity DNA polymerase (Table of Materials) are given in Supplementary Table 6.
   2. Amplify the target gene (e.g., eGFP) as a linear template from a plasmid or genome using gene-specific primers (500 nM) (for the parameters, see Supplementary Table 6).
   3. The amplification generates short extensions to provide annealing sequences for the following extension PCR steps.
   4. Check the amplicon on an agarose gel for correct size and purity.
   5. Use the amplified DNA as a template for the subsequent extension steps. Set up a reaction of at least 50 µL.
   6. Run 10 PCR amplification cycles with the extension primers (2.5 nM). After completing the amplification cycles, immediately add the final primers (500 nM) to the same reaction and run 30 cycles to amplify the extended PCR product. Find the melting temperatures and primer sequences in Supplementary Table 6.
   7. Purify the DNA fragments using a DNA purification kit and elute the DNA in nuclease-free water instead of EDTA containing elution buffer.
   8. Check the linear template on an agarose gel for correct size and purity.
   9. Measure the DNA concentration in ng/µL using an UV-Vis spectrophotometer.
2. Setting up the PURE reaction
   NOTE: The final reaction composition is 1x energy solution, tag-free ribosomes or His-tag ribosomes, OnePot PURE proteins, and DNA template. The reaction volume ratio comprises 40% energy solution, 30% protein and ribosome solution, and 30% DNA and water. Typical reaction volumes vary between 5 µL and 25 µL. Quantify the expression of a fluorescent protein continuously on a plate-reader. Use a Green Lys in vitro Translation Labeling System, which incorporates fluorescently labeled Lysine residue into newly synthesized proteins, to verify the expression of non-fluorescent proteins on a SDS-PAGE gel. An example reaction template is given in Supplementary Table 7 to help establish a PURE cell-free expression reaction. Cells in yellow indicate user-input values, and cells in orange indicate additional reagents to be optionally added to the reaction. Keep the volume ratios of the components precise to ensure the correct ion balance. For instance, to achieve a higher protein concentration, increase the OnePot protein solution concentration; however, do not increase the volume of protein solution added to the reaction.
   1. Fill in the concentration [ng/µL] and length [base pairs] of the DNA in the corresponding yellow cells in the spreadsheet. Use 2-10 nM of DNA for the reaction.
   2. Fill in the desired total reaction volume in µL.
   3. Remove the required reagents from the freezer and thaw them on ice.
      NOTE: Refreezing of the components is possible without a decrease in functionality. However, minimize the number of freeze-thaw cycles and the time samples are stored on ice as much as possible.
   4. Pipette the calculated amounts of water, DNA, and energy solution to one side of the PCR tube or one corner of a well on the 384-well plate. Add the required amount of any additional reagent on the same side. Minimize the number of samples per experiment to avoid sample evaporation and experimental start time bias.
      NOTE: It is crucial to keep the energy component physically separated from the protein components to avoid premature consumption of the energy sources and lower yields.
   5. Pipette the calculated amounts of protein and ribosome solution to the other side of a PCR tube or the opposite corner of the 384-well plate.
      NOTE: Use master mixes whenever possible to reduce the impact of pipetting errors. After initial testing, the ribosome and protein solutions can be mixed and stored as one solution.
   6. Spin for a short time (30 s) to merge the reaction components. To prevent evaporation during plate-reader experiments, add 35 µL of liquid wax and seal the plate with a transparent sealant (see Table of Materials).
   7. Incubate for a minimum of 3  h at 37 °C.
   8. For readout on a plate-reader, measure the fluorescence intensity at the required wavelength every 2 min (representative results are shown in Figure 3B).
   9. Perform the following steps for Green Lys labeled samples.
   10. After the cell-free expression, incubate the sample with 0.16 µg/µL of RNase A for 30 min at 37 °C to remove the fluorescent background of the Green Lys labeling kit.
       NOTE: Use RNase A, as other types of RNases do not remove the background sufficiently well.
   11. Visualize the protein expression by running SDS-PAGE as specified in section 1.3.3. Wash the unstained gel gently in deionized water, and image it on a fluorescent imager using an excitation wavelength of 488 nm.
   12. Subsequently, stain the gel using conventional Coomassie staining methods. For the suitable parameters see section 1.3.3.
       NOTE: Perform a titration of the protein solution with the recommended ribosome concentration and, if required, titrate ribosomes with the optimal OnePot protein concentration afterward. Use the commercial PURExpress ΔRibosome kit as a positive control. Solution A, Factor Mix, and the ribosome solution correspond to the prepared energy, the OnePot protein solution, and the purified ribosomes, respectively.

Representative Results

The above protocol is designed to facilitate establishing the PURE cell-free TX-TL system in any laboratory. The protocol includes a detailed description of the preparation of the three distinct parts of the PURE system: the OnePot protein, ribosome, and energy solution. A detailed daily schedule, which optimizes the workflow, is shown in Table 1. The workflow is optimized for the purification of His-tagged ribosomes, and time frames may differ slightly if tag-free ribosome purification is performed. One preparation provides a sufficient amount of PURE for a minimum of five hundred 10 µL reactions. Moreover, the prepared solutions are stable for more than a year at -80 °C and can withstand multiple freeze-thaw cycles.

Adequate overexpression levels for all strains are crucial for the functionality of the final protein solution. Figure 1 shows successful overexpression in all 36 individual strains used subsequently for the OnePot protein preparation. Variation in the over-expressed proteins' band intensities occurred most probably due to a bias in loading volumes onto the SDS-PAGE gel. The expected protein sizes are summarized in Table 2. GlyRS and PheRS consist of two subunits of various molecular weights; the remaining 34 proteins consist of a single subunit. Key to this protocol's simplicity and time-effectiveness is the coculturing and co-purification step (Figure 2). The OnePot protein solution was prepared by increasing the ratio of EF-Tu strain with respect to all the other expression strains. The overall composition of the final proteins was analyzed by SDS-PAGE (Figure 3A). From the gels (lanes 2, 3), it is noticeable that EF-Tu (43.3 kDa) is present in a higher concentration compared to the other proteins, as expected. While the gel provides a good first indication of protein expression ratios, it is difficult to determine whether and at which level each individual protein was expressed. Therefore, it is highly recommended to confirm the overexpression in each strain before coculturing, as shown above.

The E. coli ribosome is a complex molecular machine composed of over 50 individual protein subunits²³. A representative absorption spectrum at 260 nm for tag-free ribosome purification is shown in Figure 4; the third peak is characteristic of successful ribosome elution. For both ribosome purification methods, the expected running pattern on the SDS-PAGE gel (Figure 3A)¹⁸ was observed. We did observe contaminations for both purifications, albeit in small quantities (<10%). Notably, different contaminants were present in the tag-free (lanes 5, 6) and His-tagged (lanes 11, 12) ribosomes due to the variation in the method. For user reference, the SDS-PAGE gels for the combined systems are also included (lanes 8, 9, and 14, 15).

Lastly, the performance of the prepared systems (Figure 3) using the different ribosome variants are compared. The time courses of in vitro eGFP expression show that both PURE systems are functional and produce fluorescent eGFP. However, the OnePot protein solution combined with the His-tagged ribosomes, using the ribosome concentration optimized by titration, yielded only one-third of the expression level of the non-tagged ribosome version (Figure 3B). Similar results were observed when three proteins of different sizes were expressed and labeled using the Green Lys tRNA in vitro labeling system (Figure 3C). As seen on the fluorescent gel, full-length products were successfully expressed in both systems; however, only around half of the expression level was achieved with the His-tag ribosome system. In addition to the fluorescence labeling, the expected bands for all three proteins are distinguishable on a Coomassie-stained gel (Figure 3D). The results show that the introduced expression system, which can be prepared within a week in a laboratory with standard equipment, can be used for the in vitro expression of proteins encoded downstream of the T7 promoter from linear templates.

Figure 1: Representative results for the overexpression test for all expression strains of the PURE system. PURE protein numbers and sizes are summarized in Table 2. Protein numbers 21, 24, and 27 are marked with a star for better visualization. Please click here to view a larger version of this figure.

Figure 2: OnePot protein purification. The schematic depiction and corresponding photographs of all steps involved in the production of the OnePot protein solution. Please click here to view a larger version of this figure.

Figure 3: Performance of the prepared systems using the different ribosome variants. (A) Coomassie blue stained SDS-PAGE gels of the OnePot protein solution (lanes 2, 3), tag-free ribosomes without protein solution (lanes 5, 6) and with protein solution (lanes 8, 9), His-tagged ribosomes without protein solution (lanes 11, 12) and with protein solution (lanes 14, 15). Two different concentrations were loaded per sample. (B) Comparison of eGFP expression of His-tagged ribosomes and tag-free ribosomes. The fluorescence intensity of in vitro eGFP expression is monitored over time for a PURE reaction using tag-free ribosomes (1.8 µM, blue) and His-tagged ribosomes (0.62 µM, red). The concentrations of the linear template and the OnePot protein solution were 4 nM and 2 mg/mL, respectively. Panels (C) and (D) show the SDS-PAGE gel of proteins synthesized in OnePot with tag-free (1.8 µM, blue, lanes 3, 4, 5) and His-tag ribosomes (0.62 µM, red, lanes 6, 7, 8) labeled with a GreenLys in vitro labeling kit (C) and stained with Coomassie blue (D), respectively. The black arrows indicate the expected bands of synthesized proteins: eGFP (26.9 kDa), ArgRS (64.7 kDa), T7 RNAP (98.9 kDa). The linear template and OnePot protein solution concentrations were 4 nM and 1.6 mg/mL, respectively. Please click here to view a larger version of this figure.

Figure 4: Absorbance spectra at 260 nm. Representative results of absorbance spectra at 260 nm during hydrophobic interaction purification of tag-free ribosomes. Please click here to view a larger version of this figure.

Table 1: A daily time-optimized schedule for the preparation of all the OnePot PURE solutions. Please click here to download this Table.

Table 2: PURE protein list Please click here to download this Table.

Supplementary Table 1: Reagents. The table lists concentrations, volumes, and other specific details of the reagents and components used during this study. Please click here to download this Table.

Supplementary Table 2: Buffers. The spreadsheet lists the exact buffer compositions for protein, tag-free ribosome, and His-tag ribosome purifications, as well as the concentrations of the stock solutions used for their preparation. In addition, it calculates the required amounts of components based on the buffer volume. Please click here to download this Table.

Supplementary Table 3: Amino acid calculations. The spreadsheet lists the amino acids and their recommended stock solution concentrations required for the energy solution. It calculates the amount of water to be added to each amino acid based on the actual weighed mass, and also calculates the volume of the amino acid solution to be added to the final amino acids' mixture. Please click here to download this Table.

Supplementary Table 4: Stock solutions for the energy solution. The table lists the concentrations and volumes of stock solutions needed for the energy solution and indicates further details, including storage conditions. Please click here to download this Table.

Supplementary Table 5: Energy solution. The table lists the energy solution components and their recommended concentrations. In addition, it calculates their required volumes to be added to the final solution based on their stock solution concentrations and the volume of the energy solution. Please click here to download this Table.

Supplementary Table 6: PCR. The table lists sequences and concentrations of the primers used for the extension PCR and indicates melting temperatures and thermocycler steps optimized for a high-fidelity DNA polymerase. Please click here to download this Table.

Supplementary Table 7: PURE reaction. The spreadsheet shows an example setup of a PURE reaction. It lists the used concentrations and volumes of the components for a PURE reaction using tag-free ribosomes or His-tag ribosomes. Moreover, it calculates the volume ratios for protein and ribosome titrations. Please click here to download this Table.

Discussion

The protocol presented here describes a simple, time- and cost-effective method to prepare a versatile PURE expression system²⁰ based on the standard composition¹⁵. By utilizing the protocol together with the supplied daily schedules (Table 1), all components can be prepared in 1 week and yield amounts sufficient for up to five hundred 10 µL PURE reactions. Since the proteins used in this protocol are overexpressed from high copy plasmids and have low toxicity to E. coli, good expression levels are observed for all the required proteins (Figure 1). This allows for the easy adjustment of strains, and therefore also protein composition in cocultures, simply by modifying the ratios of the inoculation strains²⁰. Besides the ribosomal proteins, the concentration of EF-Tu showed to be of fundamental importance for expression yields⁶. In contrast, changes in the concentration of the other protein components had a relatively low impact on the robustness of the PURE system^7,24. Therefore, by adjusting the inoculation ratio of EF-Tu with regard to all the other components, a comparable composition to the standard PURE composition can be achieved, and a PURE system with a similar yield²⁰ can be attained. In preparing the protein solution, it is crucial to ensure that all strains grow well and overexpress the encoded protein after induction (Figure 1).

Ribosome function is key for the overall performance of the PURE system²⁴. In this protocol, two different methods for preparing the ribosome solution are demonstrated, i.e., tag-free and His-tagged ribosome purification. The tag-free ribosome purification is based on hydrophobic interaction chromatography followed by centrifugation with a sucrose cushion, which requires access to a FPLC purification system and an ultracentrifuge¹⁵. In contrast, the method utilizing His-tagged ribosomes¹⁸ and gravity flow affinity chromatography purification does not require specialized equipment and can be performed in most laboratories. The latter method, therefore, brings advantages such as simplicity and accessibility. However, we observed a significantly lower synthesis yield when using the His-tagged ribosomes in the OnePot PURE compared to the tag-free variant (Figure 3). Based on the type of application, this lower yield may be acceptable.

The energy solution provides the low molecular weight components and tRNAs required to fuel in vitro TX-TL reactions. This protocol provides a recipe for a typical energy solution, which can be easily adjusted based on user needs. Together with tRNA, NTP, and creatine phosphate, the abundance and concentration of Mg²⁺ ions have been crucial for the overall performance of the PURE system⁸, as they are critical cofactors for transcription and translation. In some cases, the titration of ions can, therefore, greatly enhance the overall PURE performance. DNA integrity is crucial for PURE performance. Thus, sequence verifying the promoter region, ribosome binding site, and target gene and ensuring that an adequate DNA concentration (<2 nM) will help troubleshoot issues that may arise while setting up a PURE reaction.

The PURE system is a minimal TX-TL system, and specific applications may thus require additional adjustments²⁵. These may include incorporating different RNA polymerases^9,26, chaperones¹³, and protein factors such as EF-P or ArfA⁸. Although the expression strains for these proteins can be included in the cocultures, adding them separately to the prepared system may provide better control of the required protein levels. Furthermore, the inclusion of vesicles is essential to the production of membrane proteins^10,11. Oxidizing rather than reducing environments and a disulfide bond isomerase facilitate proper disulfide bond formation, which are, for example, required for secretory proteins¹².

It is essential to ensure that any additional components do not interfere with the reaction. The most important factors to pay attention to when setting up a reaction or adding other components are listed below. Ensure that neither incompatible buffers are used nor the ion concentrations are disturbed. Avoid solutions containing glycerol, high concentrations of potassium, magnesium, calcium ions, osmolytes, pyrophosphate, antibiotics, or EDTA, as much as possible. For example, replacing an elution buffer with water during DNA purification can be beneficial as EDTA is a common additive in this buffer. Supplying the solutions with additional negatively charged molecules such as NTP or dNTP requires adjusting the magnesium concentration⁸, as the negatively charged molecules behave as chelating agents and bind positively charged molecules. A neutral pH is ideal for the reaction. Accordingly, all components should be buffered to the corresponding pH; this is especially important for highly acidic or basic molecules such as NTPs. Lastly, temperature and volume are key parameters for the reaction. To achieve a good yield, one should implement a temperature around 37 °C, as temperatures below 34 °C will significantly reduce the yield²⁷.

It is relevant to note that before preparing the OnePot PURE, one should consider the target application and the associated requirements, such as volume, purity, ease of modification, and inclusion or omission of components. For many applications, the system will be an excellent choice, but others may require yields, adjustability, and other factors, which the OnePot system cannot provide. Irrespectively, the introduced protocol will be beneficial for the preparation of any home-made system, as all critical steps for such preparation are summarized here.

One of the main advantages of the OnePot system is its compatibility with the commercially available PURExpress system, which provides the possibility of testing the functionality and integrity of all components separately by sequentially replacing each PURExpress component with its OnePot equivalent. The advantages of the OnePot PURE system, such as tunability and easy, fast, and cost-effective preparation, will make cell-free TX-TL accessible to more laboratories worldwide and contribute to expanding the implementation of this powerful platform in cell-free synthetic biology.

Materials

Name	Company	Catalog Number	Comments
10x Tris/Glycine/SDS buffer	Bio-Rad Laboratories	1610732
15 mL centrifuge tubes	VWR International	525-0309
384-well Black Assay Plates	Corning	3544
4-20% Mini-PROTEANRTM TGXTM Precast Protein Gels	Bio-Rad Laboratories	4561096
50 mL centrifuge tubes	VWR International	525-0304
96-Well Polypropylene DeepWell plate	Nunc	260252
Acetic acid, 99.8 %	Acros	222140010
Äkta purifier	GE Healthcare		purification of tag free ribosomes
AMICON ULTRA 0.5 mL - 3 KDa	Merck Millipore	UFC500324
AMICON ULTRA 15 mL - 3 KDa	Merck Millipore	UFC900324
Amino acids	Sigma-Aldrich	LAA21-1KT
Ammonium chloride	Sigma-Aldrich	09718-250G
Ammonium sulfate	Sigma-Aldrich	A4418
Ampicillin	Condalab	6801
BenchMark Fluorescent Protein Standard	ThermoFisher	LC5928
Breathe-Easy sealing membrane	Diversified Biotech	Z380059-1PAK
Centrifuge tubes polycarbonate	Beckman	355631	purification of tag free ribosomes
Chill-out Liquid Wax	Bio-Rad Laboratories	CHO1411
Creatine phosphate	Sigma-Aldrich	27920
DNA Clean & Concentrator-25 (Capped)	Zymo	ZYM-D4034-200TS
DTT	SantaCruz Biotech	sc-29089B
Econo-Pac Chromatography Columns	Bio-Rad Laboratories	7321010
EDTA (Ethylenediaminetetraacetic acid)	Sigma-Aldrich	03609-250G
Eppendorf Protein LoBind microcentrifuge tubes	VWR International / Eppendorf	525-0133
Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap	Falcon	352051
Flasks, baffled 1000 mL 4 baffles, borosilicate glass	Scilabware	9141173
FluoroTect Green Lys in vitro Translation Labeling System	Promega	L5001	optional
Folinic acid	Sigma-Aldrich	PHR1541
Glycerol	Sigma-Aldrich	G7757-1L
HEPES	Gibco	15630-056
HiTrap Butyl HP Column	GE Healthcare	28411005	purification of tag free ribosomes
IMAC Sepharose 6 Fast Flow	GE Healthcare	17-0921-07
Imidazole	Sigma-Aldrich	I2399
InstantBlue	Expedeon	ISB1L-1L
IPTG (Isopropyl-beta-D-thiogalactoside)	Alfa Aesar	B21149.03
Laemmli buffer (2x), sample buffer	Sigma-Aldrich	S3401-1VL
Lysogeny broth (LB) media	AppliChem	A0954
Magnesium acetate	Sigma-Aldrich	M0631
Magnesium chloride	Honeywell Fluka	63020-1L
Nickel Sulfate	Alfa Aesar	15414469
NTP	ThermoFisher	R0481
Phusion High-Fidelity DNA Polymerase (2 U/µL)	ThermoFisher	F530S
Potassium chloride	Sigma-Aldrich	P5405-1KG
Potassium glutamate	Sigma-Aldrich	49601
PURExpress In Vitro Protein Synthesis Kit	NEB	E6800S
PURExpress Δ Ribosome Kit	NEB	E3313S
Quick Start Bradford 1x Dye Reagent	Bio-Rad Laboratories	5000205
Rapid-Flow Sterile Single Use Vacuum Filter Units	ThermoFisher	564-0020
RNaseA solution	Promega	A7973
SealPlate film	Excel Scientific	Z369659-100EA
Sodium hydroxide	Sigma-Aldrich	6203
Spermidine	Sigma-Aldrich	S2626
Sucrose	Sigma-Aldrich	84097
TCEP (Tris(2-carboxyethyl)phosphin -hydrochlorid)	Sigma-Aldrich	646547-10X1mL
Thickwall Polycarbonate Tube	Beckman	355631
Trichloroacetic acid	Sigma-Aldrich	T0699
Tris base	ThermoFisher	BP152-500
tRNA	Roche	10109541001
Ultracentrifuge Optima L-80	Beckman		purification of tag free ribosomes
Whatman GD/X syringe filters	GE Whatman	WHA68722504
β-mercaptoethanol	Sigma-Aldrich	M6250-100mL