Guide Cell-Free Protein Synthesis: Methods and Protocols

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Depicted are the biomass solid line and the differential specific growth rate dashed line. For lysate preparation, cells were harvested at maximum growth. Error bars denote standard deviations of three independent measurements. The negative control without plasmid is also shown dashed line. The biomass was harvested at maximum growth after only 2 h of cultivation and cell-free extract was prepared as described earlier in the text. To test the potential of in vitro translation for the cell-free extract from V. We used the identical reaction setup as described in our previous report for E.

Concentrations of substrates amino acids and nucleotides , salts, and buffer components were also adopted. Exogenous energy regeneration by creatine phosphate and creatine kinase was used to supply the system with regenerated ATP and GTP. Notably, combined transcription and translation in the V. Expression of eGFP was detected after 30 min of incubation and was accompanied by a linear increase of fluorescence over the course of about 1 h. Active synthesis was followed by a decline of the synthesis rate and a termination of the reaction after a total reaction time of 2 h.

Upon demonstration of active protein synthesis by a cell-free translation system based on V. As ribosomes denote the key for translation, we aimed to quantify the rRNA concentration in the extract. This concentration is roughly four-times higher than that in our standard E. Moreover, the measured rRNA concentration correlates nicely with the approximately four-fold higher specific growth rate between V.

In addition, as we always resuspended 1 g biomass wet weight in 1 mL lysis buffer prior to homogenization, the direct comparison of certain characteristics, such as the ribosome concentration between both systems, is justified. CFPS depends on highly active catalytic proteins provided by the cell-free extract. However, although we have previously shown that the cell-free extract preparation procedure preserves crucial components involved in translation in the case of E. Hence, we concluded that similar to E. Supplying the transcription and translation reactions with energy is crucial for efficient protein synthesis Kim and Swartz, ; Kim and Kim, Therefore, we compared several of the most common energy regeneration systems, namely creatine phosphate in combination with creatine kinase, phosphoenolpyruvate PEP , and pyruvate, for their ability to energize the translation reaction in the V.

For each system, we optimized the magnesium ion concentration, a parameter well known to impact the translation performance Kim and Choi, ; Failmezger et al. Similar to cell-free translation systems from other source organisms; e. This is demonstrated in Figure 2 , which illustrates the relative synthesis rate at different concentrations of magnesium at a constant concentration of 60 mM creatine phosphate. A clear optimum at 18 mM magnesium was achieved. Cell-free protein synthesis energized by creatine phosphate and creatine kinase. Differential rate of eGFP synthesis over time at different magnesium concentrations.

Error bars represent standard deviations of triplicate measurements. Next, we investigated the potential of endogenous energy regeneration by supplying the reaction with PEP or pyruvate. A similar scenario was obtained using pyruvate to regenerate ATP, with similar final titers and maximum synthesis rates also being obtained Supplementary Figure S2B. Thus, it can be concluded that whereas energy regeneration by endogenous enzymes in the V. This may be due to the high activity of metabolic pathways unfavorably channeling glycolytic substrates away from ATP synthesis.

Our finding is similar to that observed in other cell-free systems, such as upon blocking the conversion of pyruvate to PEP by phosphoenolpyruvate synthetase using oxalate increased productivity in an E. Whether a similar increase in productivity can be achieved in the V. The supply of the translation reaction with amino acids has previously been shown to be critical in cell-free systems Kim and Choi, ; Jewett and Swartz, b ; Calhoun and Swartz, ; Kim et al.

A bottleneck in the amino acid supply can partially be overcome by raising the amino acids concentration up to 2 mM Kim and Swartz, or by designing strains lacking several amino acid degradation pathways Calhoun and Swartz, As it can be assumed that amino acid degradation is prevalent in our reaction, we investigated the amino acid stability in the CFPS system from V. Therefore, samples were taken after 3 h of reaction time and the amino acids content was determined via high performance liquid chromatography HPLC analysis and compared to initial conditions.

Methods and Protocols

In the reaction system empowered by creatine phosphate, the three amino acids aspartic acid, serine, and arginine were almost entirely consumed Figure 3A. Notably, aspartic acid was entirely depleted in the reaction, whereas more than half of the amounts of serine and arginine were degraded. A similar scenario was obtained for the system fueled by PEP, wherein it was also demonstrated that aspartic acid, serine, and arginine could be regarded as most critical Figure 3B.

Moreover, the amount of alanine doubled in the reaction fueled by PEP. It may be hypothesized that several enzymatic activities are responsible for the observed turnover of amino acids. For example, it was previously demonstrated that arginine depletion was due to arginine decarboxylase activity in a CFPS system from E. In addition, it was claimed that the decrease of serine is associated with pyruvate formation by serine deaminase Calhoun and Swartz, It is therefore likely that similar enzymatic activities are present in the cell-free extract from V.

Amino acid degradation in the V. Initial concentrations were set to 1 mM of each amino acid. Note that cysteine could not be reliably measured using our analysis method. For transcription of eGFP we used our standard expression plasmid, which we prepared from E. However, to test whether the plasmid source affected the transcriptional and translational performance, we purified the plasmid from V. As depicted in Figure 4 , the source of the plasmid markedly affected the translation performance. Specifically, the V. Notably, the control experiment using our established E.

It can be hypothesized that differences in the restriction-modification system between both species impact the plasmid stability and subsequently the transcription and translation performance. This is emphasized by the fact that the transformation efficiency of V. Therefore, it could be concluded that it is essential that the expression plasmid have the same cellular background as the CFPS systems to which it is applied.

CFPS performed with V. The same experiment was also performed with E. The maximum eGFP concentration obtained after 3 h of an in vitro translation reaction is shown. Plasmid concentrations were kept constant for all experiments. Therefore, we chose to test whether a similar transcription unit could be activated in our V. Considering the fundamental similarities between the two systems described above, we reasoned that this promoter sequence could also function in the V.

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However, we considered that the use of core RNAP might lead to extended synchronization of transcription and translation events, thus enabling more efficient translation in earlier reaction times, as was observed. Overall, this experiment clearly demonstrated that the V. This motivated us to evaluate the rRNA stability in the V. In contrast, providing magnesium homeostasis by using pyruvate resulted in elevated stability of both rRNA species Figure 6B. Moreover, the electropherograms revealed no cleavage products Supplementary Figure S3 ; therefore, it could be hypothesized that the rRNA degradation can be characterized by an exonuclease activity.

Thus, our efforts clearly demonstrated that magnesium also constitutes a key parameter for efficient translation and preservation of ribosome integrity in the V. A CFPS fueled with creatine phosphate, which results in the accumulation of phosphate and magnesium sequestration. Using the insights obtained regarding the V.

This included, e. Therefore, by scaling down the reaction, final product titers could be further increased up to four-fold Figure 7B. From this experiment, it was clear that the performance levels achieved thus far did not meet the expectations with respect to the measured high ribosome concentration in the extract.

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Although bulk protein synthesis rates for published E. Consequently, owing to the observed still low volumetric synthesis rates, it might be inferred that only a partial fraction of the ribosomes could be activated under the current reaction conditions. Therefore, further strategies to improve the performance and to unleash the theoretical power of the system needed to be developed. In this regard, following the already established guiding principle of advancing CFPS by cytoplasmic mimicry Jewett and Swartz, a ; Jewett et al. Notably, recent reports propose a similar scenario for in vitro translation systems from E.

Hence, it might be suggested that limited ribosome activity represents a general bottleneck in CFPS. A valuable application of CFPS systems is the rapid screening for protein expression using different regulation entities Sawasaki et al. A step toward this goal is the identification of strong promoters to allow efficient gene expression.

To demonstrate the potential of our developed CFPS system to enable the identification of possible promoters of V. Although little is known regarding promoters from V. Lacking any information of comparable weak promoters, we relied upon the weak promoter P lacI from E. Screening this small library showed three distinct output signals Figure 8. Whereas the highest eGFP expression was obtained for the strong V. This experiment clearly demonstrated the ability to screen for promoter strength in a straightforward and rapid manner using our established CFPS system from V.

Herein, we reported the development of a V.


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Thorough characterization revealed the enormous potential of this system to synthesize proteins based on the measured concentration of rRNA in the cell-free extract. However, further improvements of the reaction setup and the screening of factors currently limiting the overall performance of the system, e. Towards this end, the application of Vibrio specific components such as tRNA, a detailed component optimization beyond E.

Whereas these limitations will be addressed in future work, the applicability of our V. Looking forward, we believe that our study represents the first step toward the establishment of a high yielding CFPS system based on extract from V. During the cultivation, samples were taken and cell density was determined at nm using a photometer. When the exponential phase was reached the biomass was rapidly chilled by incubation in an ice bath. Cell free lysate of V. The frozen biomass was thawed in 1 mL cold S30 buffer per g biomass [14 mM magnesium acetate, 60 mM potassium acetate, 10 mM Tris, pH 8.


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The cell suspension was lysed by passage through a high-pressure homogenizer Emulsi-Flex-C5, Avestin, Canada at 12, kPa. The cell-free extract was then dialyzed against a fold larger volume of S30 dialysis buffer 14 mM magnesium acetate, 60 mM potassium acetate, 10 mM Tris, pH 8. Prior to each reading cycle the plates were shaken. The extraction of the total RNA and the analysis of the rRNA from the cell lysate using capillary gel electrophoresis with laser-induced fluorescence detection was performed as described previously Failmezger et al.

To quantify amino acids during the cell free reaction, samples were taken after 0, 90, and min. Before injection, the amino acids were automatically pre-column derivatized with ortho-phthaldialdehyde and fluorenylmethoxycarbonyl chloride. The following gradient was generated at a flow rate of 1. Detection of the derivatized L -amino acids occurred via a fluorescence detector, with different retention times corresponding to the single derivatized L -amino acids.

The L -amino acids were quantified using an 8-point calibration curve for each amino acid as an external reference standard and by using L -ornithine as an internal standard. The Plasmid pJOE The plasmids pJOE All three plasmids contain an ampR marker for selection. The promoter sequences were amplified from genomic DNA of V. The plasmids were transformed into V. JF designed the study, analyzed the data, wrote and drafted the manuscript. SS performed the experiments and analyzed the data. MS-H was responsible for this study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Aiyar, S. Anastasina, M. Active synthesis was followed by a decline of the synthesis rate and a termination of the reaction after a total reaction time of 2 h. Upon demonstration of active protein synthesis by a cell-free translation system based on V.

As ribosomes denote the key for translation, we aimed to quantify the rRNA concentration in the extract. This concentration is roughly four-times higher than that in our standard E. Moreover, the measured rRNA concentration correlates nicely with the approximately four-fold higher specific growth rate between V. In addition, as we always resuspended 1 g biomass wet weight in 1 mL lysis buffer prior to homogenization, the direct comparison of certain characteristics, such as the ribosome concentration between both systems, is justified. CFPS depends on highly active catalytic proteins provided by the cell-free extract.

However, although we have previously shown that the cell-free extract preparation procedure preserves crucial components involved in translation in the case of E. Hence, we concluded that similar to E. Supplying the transcription and translation reactions with energy is crucial for efficient protein synthesis Kim and Swartz, ; Kim and Kim, Therefore, we compared several of the most common energy regeneration systems, namely creatine phosphate in combination with creatine kinase, phosphoenolpyruvate PEP , and pyruvate, for their ability to energize the translation reaction in the V.

For each system, we optimized the magnesium ion concentration, a parameter well known to impact the translation performance Kim and Choi, ; Failmezger et al. Similar to cell-free translation systems from other source organisms; e. This is demonstrated in Figure 2 , which illustrates the relative synthesis rate at different concentrations of magnesium at a constant concentration of 60 mM creatine phosphate. A clear optimum at 18 mM magnesium was achieved. Cell-free protein synthesis energized by creatine phosphate and creatine kinase. Differential rate of eGFP synthesis over time at different magnesium concentrations.

Error bars represent standard deviations of triplicate measurements. Next, we investigated the potential of endogenous energy regeneration by supplying the reaction with PEP or pyruvate. A similar scenario was obtained using pyruvate to regenerate ATP, with similar final titers and maximum synthesis rates also being obtained Supplementary Figure S2B. Thus, it can be concluded that whereas energy regeneration by endogenous enzymes in the V. This may be due to the high activity of metabolic pathways unfavorably channeling glycolytic substrates away from ATP synthesis.

Cell-free protein synthesis - Wikipedia

Our finding is similar to that observed in other cell-free systems, such as upon blocking the conversion of pyruvate to PEP by phosphoenolpyruvate synthetase using oxalate increased productivity in an E. Whether a similar increase in productivity can be achieved in the V. The supply of the translation reaction with amino acids has previously been shown to be critical in cell-free systems Kim and Choi, ; Jewett and Swartz, b ; Calhoun and Swartz, ; Kim et al.

A bottleneck in the amino acid supply can partially be overcome by raising the amino acids concentration up to 2 mM Kim and Swartz, or by designing strains lacking several amino acid degradation pathways Calhoun and Swartz, As it can be assumed that amino acid degradation is prevalent in our reaction, we investigated the amino acid stability in the CFPS system from V. Therefore, samples were taken after 3 h of reaction time and the amino acids content was determined via high performance liquid chromatography HPLC analysis and compared to initial conditions.

In the reaction system empowered by creatine phosphate, the three amino acids aspartic acid, serine, and arginine were almost entirely consumed Figure 3A. Notably, aspartic acid was entirely depleted in the reaction, whereas more than half of the amounts of serine and arginine were degraded. A similar scenario was obtained for the system fueled by PEP, wherein it was also demonstrated that aspartic acid, serine, and arginine could be regarded as most critical Figure 3B.

Moreover, the amount of alanine doubled in the reaction fueled by PEP. It may be hypothesized that several enzymatic activities are responsible for the observed turnover of amino acids. For example, it was previously demonstrated that arginine depletion was due to arginine decarboxylase activity in a CFPS system from E. In addition, it was claimed that the decrease of serine is associated with pyruvate formation by serine deaminase Calhoun and Swartz, It is therefore likely that similar enzymatic activities are present in the cell-free extract from V.

Amino acid degradation in the V. Initial concentrations were set to 1 mM of each amino acid. Note that cysteine could not be reliably measured using our analysis method.

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For transcription of eGFP we used our standard expression plasmid, which we prepared from E. However, to test whether the plasmid source affected the transcriptional and translational performance, we purified the plasmid from V. As depicted in Figure 4 , the source of the plasmid markedly affected the translation performance. Specifically, the V. Notably, the control experiment using our established E. It can be hypothesized that differences in the restriction-modification system between both species impact the plasmid stability and subsequently the transcription and translation performance.

This is emphasized by the fact that the transformation efficiency of V. Therefore, it could be concluded that it is essential that the expression plasmid have the same cellular background as the CFPS systems to which it is applied. CFPS performed with V. The same experiment was also performed with E. The maximum eGFP concentration obtained after 3 h of an in vitro translation reaction is shown. Plasmid concentrations were kept constant for all experiments. Therefore, we chose to test whether a similar transcription unit could be activated in our V.

Considering the fundamental similarities between the two systems described above, we reasoned that this promoter sequence could also function in the V. However, we considered that the use of core RNAP might lead to extended synchronization of transcription and translation events, thus enabling more efficient translation in earlier reaction times, as was observed. Overall, this experiment clearly demonstrated that the V. This motivated us to evaluate the rRNA stability in the V. In contrast, providing magnesium homeostasis by using pyruvate resulted in elevated stability of both rRNA species Figure 6B.

Moreover, the electropherograms revealed no cleavage products Supplementary Figure S3 ; therefore, it could be hypothesized that the rRNA degradation can be characterized by an exonuclease activity. Thus, our efforts clearly demonstrated that magnesium also constitutes a key parameter for efficient translation and preservation of ribosome integrity in the V. A CFPS fueled with creatine phosphate, which results in the accumulation of phosphate and magnesium sequestration.

Using the insights obtained regarding the V. This included, e. Therefore, by scaling down the reaction, final product titers could be further increased up to four-fold Figure 7B. From this experiment, it was clear that the performance levels achieved thus far did not meet the expectations with respect to the measured high ribosome concentration in the extract.

Although bulk protein synthesis rates for published E. Consequently, owing to the observed still low volumetric synthesis rates, it might be inferred that only a partial fraction of the ribosomes could be activated under the current reaction conditions. Therefore, further strategies to improve the performance and to unleash the theoretical power of the system needed to be developed.

In this regard, following the already established guiding principle of advancing CFPS by cytoplasmic mimicry Jewett and Swartz, a ; Jewett et al. Notably, recent reports propose a similar scenario for in vitro translation systems from E. Hence, it might be suggested that limited ribosome activity represents a general bottleneck in CFPS.

A valuable application of CFPS systems is the rapid screening for protein expression using different regulation entities Sawasaki et al. A step toward this goal is the identification of strong promoters to allow efficient gene expression. To demonstrate the potential of our developed CFPS system to enable the identification of possible promoters of V.

Although little is known regarding promoters from V. Lacking any information of comparable weak promoters, we relied upon the weak promoter P lacI from E. Screening this small library showed three distinct output signals Figure 8. Whereas the highest eGFP expression was obtained for the strong V. This experiment clearly demonstrated the ability to screen for promoter strength in a straightforward and rapid manner using our established CFPS system from V.

Herein, we reported the development of a V. Thorough characterization revealed the enormous potential of this system to synthesize proteins based on the measured concentration of rRNA in the cell-free extract. However, further improvements of the reaction setup and the screening of factors currently limiting the overall performance of the system, e. Towards this end, the application of Vibrio specific components such as tRNA, a detailed component optimization beyond E.

Whereas these limitations will be addressed in future work, the applicability of our V. Looking forward, we believe that our study represents the first step toward the establishment of a high yielding CFPS system based on extract from V. During the cultivation, samples were taken and cell density was determined at nm using a photometer. When the exponential phase was reached the biomass was rapidly chilled by incubation in an ice bath. Cell free lysate of V.

The frozen biomass was thawed in 1 mL cold S30 buffer per g biomass [14 mM magnesium acetate, 60 mM potassium acetate, 10 mM Tris, pH 8. The cell suspension was lysed by passage through a high-pressure homogenizer Emulsi-Flex-C5, Avestin, Canada at 12, kPa. The cell-free extract was then dialyzed against a fold larger volume of S30 dialysis buffer 14 mM magnesium acetate, 60 mM potassium acetate, 10 mM Tris, pH 8. Prior to each reading cycle the plates were shaken. The extraction of the total RNA and the analysis of the rRNA from the cell lysate using capillary gel electrophoresis with laser-induced fluorescence detection was performed as described previously Failmezger et al.

To quantify amino acids during the cell free reaction, samples were taken after 0, 90, and min. Before injection, the amino acids were automatically pre-column derivatized with ortho-phthaldialdehyde and fluorenylmethoxycarbonyl chloride. The following gradient was generated at a flow rate of 1. Detection of the derivatized L -amino acids occurred via a fluorescence detector, with different retention times corresponding to the single derivatized L -amino acids.

The L -amino acids were quantified using an 8-point calibration curve for each amino acid as an external reference standard and by using L -ornithine as an internal standard. The Plasmid pJOE The plasmids pJOE All three plasmids contain an ampR marker for selection. The promoter sequences were amplified from genomic DNA of V.

The plasmids were transformed into V. JF designed the study, analyzed the data, wrote and drafted the manuscript. SS performed the experiments and analyzed the data. MS-H was responsible for this study. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Aiyar, S. Anastasina, M. Biotechniques 56, 36— Google Scholar. Cell-free protein expression based on extracts from CHO cells. Calhoun, K. Total amino acid stabilization during cell-free protein synthesis reactions. Cordova, L. Complete genome sequence, metabolic model construction and phenotypic characterization of geobacillus lc, an extremely thermophilic, fast growing, xylose-utilizing bacterium. Eagon, R. Pseudomonas natriegens , a marine bacterium with a generation time of less than 10 minutes.

PubMed Abstract Google Scholar. Failmezger, J.