Continuous cell-free protein synthesis using glycolytic intermediates as energy sources

In this work, we demonstrate that glycolytic intermediates can serve as efficient energy sources to regenerate ATP during continuous-exchange cell-free (CECF) protein synthesis reactions. Through the use of an optimal energy source, approximately 10 mg/ml of protein was generated from CECF protein synthesis reaction at greatly reduced reagent costs. Compared with the conventional reactions utilizing phosphoenol pyruvate as an energy source, the described method yields 10-fold higher productivity per unit reagent cost, making the techniques of CECF protein synthesis more realistic alternative for rapid protein production.     

An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates

Cell-free protein synthesis reactions have not been seriously considered as a viable method for commercial protein production mainly because of high reagent costs and a lack of scalable technologies. Here we address the first issue by presenting a cell-free protein synthesis system with comparable protein yields that removes the most expensive substrates and lowers the cell-free reagent cost by over 75% (excluding extract, polymerase, and plasmid) while maintaining high energy levels. This system uses glucose as the energy source and nucleoside monophosphates (NMPs) in place of nucleoside triphosphates (NTPs) as the nucleotide source. High levels of nucleoside triphosphates are generated from the monophosphates within 20 min, and the subsequent energy charge is similar in reactions beginning with either NTPs or NMPs. Furthermore, significant levels (>0.2 mM) of all NTPs are still available at the end of a 3-h incubation, and the total nucleotide pool is stable throughout the reaction. The glucose/NMP reaction was scaled up to milliliter scale using a thin film approach. Significant yields of active protein were observed for two proteins of vastly different size: chloramphenicol acetyl transferase (CAT, 25 kDa) and beta-galactosidase (472 kDa). The glucose/NMP cell-free reaction system dramatically reduces reagent costs while supplying high protein yields.     

Energizing cell-free protein synthesis with glucose metabolism

In traditional cell-free protein synthesis reactions, the energy source (typically phosphoenolpyruvate (PEP) or creatine phosphate) is the most expensive substrate. However, for most biotechnology applications glucose is the preferred commercial substrate. Previous attempts to use glucose in cell-free protein synthesis reactions have been unsuccessful. We have now developed a cell-free protein synthesis reaction where PEP is replaced by either glucose or glucose-6-phosphate (G6P) as the energy source, thus allowing these reactions to compete more effectively with in vivo protein production technologies. We demonstrate high protein yields in a simple batch-format reaction through pH control and alleviation of phosphate limitation. G6P reactions can produce high protein levels ( approximately 700 microg/mL of chloramphenical acetyl transferase (CAT)) when pH is stabilized through replacement of the HEPES buffer with Bis-Tris. Protein synthesis with glucose as an energy source is also possible, and CAT yields of approximately 550 mug/mL are seen when both 10 mM phosphate is added to alleviate phosphate limitations and the Bis-Tris buffer concentration is increased to stabilize pH. By following radioactivity from [U-(14)C]-glucose, we find that glucose is primarily metabolized to the anaerobic products, acetate and lactate. The ability to use glucose as an energy source in cell-free reactions is important not only for inexpensive ATP generation during protein synthesis, but also as an example of how complex biological systems can be understood and exploited through cell-free biology.     

Reconstitution of leucine-mediated autophagy via the mTORC1-Barkor pathway in vitro

Supplementation of branched chain amino acids, especially leucine, is critical to improve malnutrition by regulating protein synthesis and degradation. Emerging evidence has linked leucine deprivation induced protein breakdown to autophagy. In this study, we aimed to establish a cell-free assay recapitulating leucine-mediated autophagy in vitro and dissect its biochemical requirement. We found that in a cell-free assay, membrane association of Barkor/Atg14(L), a specific autophagosome-binding protein, is suppressed by cytosol from nutrient-rich medium and such suppression is released by nutrient deprivation. We also showed that rapamycin could efficiently reverse the suppression of nutrient rich cytosol, suggesting an essential role of mTORC1 in autophagy inhibition in this cell-free system. Furthermore, we demonstrated that leucine supplementation in the cultured cells blocks Barkor puncta formation and autophagy activity. Hence, we establish a novel cell-free assay recapitulating leucine-mediated autophagy inhibition in an mTORC1-dependent manner; this assay will help us to dissect the regulation of amino acids in autophagy and related human metabolic diseases.     

Effect of 1,4-dihydroxy-5,8-bis (2-(2-hydroxyethylamino) ethyl amino)-9,10-anthracenedione (dihydroxyanthraquinone) on the replication of simian virus 40 chromosome

The effect of dihydroxyanthraquinone on mammalian chromosome structure and replication was investigated using simian virus 40 chromosome as a model system. Viral DNA synthesis in African green monkey kidney cells was approximately 90% inhibited by the drug at 0.4 μM. RNA or protein synthesis was inhibited only 50% under the same conditions. Both single-stranded and double-stranded breakage of viral DNA were found on viral chromosomes isolated from infected cells treated with the drug. Four distinct viral chromosomal templates were found in nuclear extract prepared from cells treated with the drug as determined by the cell-free system for viral DNA synthesis. The results suggested that dihydroxyanthraquinone acts at the level of chromosome replication.     

Development of a rapid and productive cell-free protein synthesis system

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast toin vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately 650 μg/mL of protein was produced after 2 h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.     

Cell-free protein synthesis system prepared from insect cells by freeze-thawing

We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and beta-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5'-untranslated region (5'-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5'-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5'-UTRs tested. As a result, in a batch reaction approximately 71 microg of luciferase was synthesized per milliliter of reaction volume at 25 degrees C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5'-UTR of mRNA, approximately 45 microg/mL of luciferase was synthesized in an Sf21 cell-free system at 25 degrees C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method.     

Development of an E. coli strain for cell-free ADC manufacturing

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody–drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.     

Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system

We developed a cell-free protein synthesis system that produces more than 1mg/ml of recombinant proteins in two hours. A basal system that supports the stable maintenance of ATP and amino acids was constructed by using high concentrations of CP (100 mM) and amino acids (3 mM). Approximately 0.6 mg/ml of protein was produced during the batch incubation of the basal system. We found that the accumulation of inorganic phosphate reduces the concentration of free magnesium ions and that there exists a critical concentration of magnesium at which the protein synthesis is halted. Based on this finding, we attempted to extend the duration of the protein synthesis by keeping the magnesium concentration sufficiently high throughout the reaction period. The protein synthesis reaction continued for at least 2 h when the reaction was repeatedly supplemented with magnesium, and approximately 1.2 mg/ml of active CAT or GFP was produced. The simple, fast, and highly productive cell-free protein synthesis system described herein should offer a versatile platform for the preparation of protein molecules in various post-genomic efforts.     

The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract

In our experiments to produce different combinations of recombinant proteins in a cell-free protein synthesis system derived from Escherichia coli, we found that certain pairs of ORFs were not expressed equally. Instead, only a single DNA species was expressed dominantly, while the expression of the others was almost completely repressed. This bias during the co-expression of the DNA pairs was eliminated when an identical downstream box sequence was added to the 5'-ends of the template DNA pairs. By introducing identical nucleotide sequences of the his-tag or the downstream box of chloramphenicol acetyltransferase (CAT-DB) in front of the target genes that were otherwise not expressed compatibly, both of the encoded proteins were produced at similar productivities. Moreover, in the presence of a common downstream box, multiple genes were simultaneously expressed in the same reaction mixture. We expect that the proposed approach will offer a powerful tool for the preparation of unbiased protein libraries, as well as for studying the structure and functions of interacting proteins.     

A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery

Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms.     

Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.     

Cell-free production of aggregation-prone proteins in soluble and active forms

We have developed an efficient cell-free protein synthesis system for the production of soluble and active eukaryotic proteins that are predominantly produced as inclusion bodies in bacteria. S30 extracts (indicating the supernatant of cell homogenate when centrifuged at 30,000g) for cell-free protein synthesis were prepared from Escherichia coli that was modified to overexpress a set of chaperones (GroEL/ES or DnaK/J-GrpE) and disulfide isomerase (leader sequence-free mature DsbC expressed in the cytoplasm). The solubility and biological activity concentration (biological activity per unit volume of cell-free protein synthesis reaction mixture) of the protein synthesized by the new cell-free protein synthesis system showed a dramatic improvement. Solubility enhancement was most dramatic with the existence of DnaK/J-GrpE. It shows that the co-translational interaction with DnaK/J-GrpE prior to folding trial is important in maintenance of the aggregation-prone protein in a folding-competent soluble state. For maximizing the biological activity concentration of the expressed protein, the additional presence of GroEL/ES and DsbC was required. When human erythropoietin was expressed in the developed cell-free protein synthesis system including endogenously overexpressed chaperones and/or DsbC, the biological activity concentration of erythropoietin was enhanced by 700%. It implies that the post-translational folding and disulfide bond reshuffling as well as co-translational folding are important in acquiring functionally active protein from cell-free expression system. This is the first report of using S30 extracts including endogenously overexpressed chaperones and/or disulfide isomerase for the efficient production of soluble and active proteins in cell-free protein synthesis. This new cell-free protein synthesis system was capable of introducing much larger amounts of chaperones and disulfide isomerase compared to a conventional method that supplements them separately. The developed cell-free protein synthesis system supported efficient expression of the eukaryotic proteins in soluble and active forms without the need of any exogenous addition or coexpression of folding effectors.     

Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system

In this study, as a part of our efforts to improve the robustness and economical feasibility of cell-free protein synthesis, we developed a simple method of preparing the cell extracts used for catalyzing cell-free protein synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support cell-free protein synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.     

Partial characterizatio and proposed mode of action of inhibitory heLa cell surface polypeptides

Polypeptides removed from the HeLa cell surface by mild pronase treatment rapidly inhibit protein synthesis when added to HeLa cells or cell-free translation system derived from HeLa cells. The inhibitory activity is heat stable. Protein and carbohydrate components of these polypeptides are required for inhibition of protein synthesis in vivo and in vitro. Two peaks of activity can be recovered from polyacrylamide gels, corresponding to polypeptides with molecular weights of approximately 29 000 and 41 000. Inhibition of protein synthesis in cell-free translation systems appears to be primarily an effect on elongation of polypeptide chains, whereas in the intact cell the primary target may be polypeptide chain initiation.     

Effects of growth rate on cell extract performance in cell-free protein synthesis

Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity.     

A tentative model of formation of structural proteins of tick-borne encephalitis virus (flavivirus)

The time course of tick-borne encephalitis virus cell-free protein synthesis was studied by using either [³⁵S]-methionine or formyl[³⁵S]methionyl-tRNA_f^Met as substrates, and the [³⁵S]methionine-labelled products were compared by fingerprinting tryptic peptides. An intermediate in the protein processing, the polypeptide doublet p36/33, was characterized and a tentative model for flavivirus structural protein synthesis and processing was proposed.     

A bilayer cell-free protein synthesis system for high-throughput screening of gene products

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.