Review Article | Open Access
Yi Cui, Xinjie Chen, Ze Wang, Yuan Lu, "Cell-Free PURE System: Evolution and Achievements", BioDesign Research, vol. 2022, Article ID 9847014, 11 pages, 2022. https://doi.org/10.34133/2022/9847014
Cell-Free PURE System: Evolution and Achievements
The cell-free protein synthesis (CFPS) system, as a technical core of synthetic biology, can simulate the transcription and translation process in an in vitro open environment without a complete living cell. It has been widely used in basic and applied research fields because of its advanced engineering features in flexibility and controllability. Compared to a typical crude extract-based CFPS system, due to defined and customizable components and lacking protein-degrading enzymes, the protein synthesis using recombinant elements (PURE) system draws great attention. This review first discusses the elemental composition of the PURE system. Then, the design and preparation of functional proteins for the PURE system, especially the critical ribosome, were examined. Furthermore, we trace the evolving development of the PURE system in versatile areas, including prototyping, synthesis of unnatural proteins, peptides and complex proteins, and biosensors. Finally, as a state-of-the-art engineering strategy, this review analyzes the opportunities and challenges faced by the PURE system in future scientific research and diverse applications.
The purpose of cell-free synthetic biology is to understand, utilize, and expand the functions of natural biological systems without using the whole living cells. It breaks the boundary between nonliving chemicals and living bodies. Through purposeful design, transformation, and resynthesis of living systems, it even creates and endows “artificial life” with unnatural functions, thereby promoting innovation from mimicking life to recreating life [1–4]. The cell-free protein synthesis (CFPS) system is the core technology of cell-free synthesis biology , also known as in vitro protein transcription and translation technology.
Since the first successful construction of the CFPS system by Nirenberg and Matthaei , after nearly 70 years of research, people have found that the CFPS system has advantages in operation and application compared with the traditional cell system. In terms of operation, the CFPS system, as an in vitro life simulation system, avoids the tedious gene cloning and cell culture operations in the process of protein synthesis in the cell system. The CFPS system bypasses the cell wall, eliminates gene regulation, and can synthesize a large number of proteins in a short time. In addition to operability, the CFPS system has four advantages compared with the traditional cell system in application: safety, tolerance, storability, and fast response. First of all, CFPS does not involve live artificial transgenic cells, avoiding the risk of cell replication and transmission, so there is no biosafety problem . Second, the CFPS system can operate in the presence of toxins , so it is tolerant to various chemical or biological agents. Third, through freeze-drying technology, the CFPS system can be stored stably for a long time . Finally, its open environment avoids the obstacles of material transmembrane transport, so that the substance to be tested can be in direct contact with the reaction system, with shorter reaction time, higher sensitivity, and wider detection range. After years of development, modern CFPS systems can be divided into two types, including complex crude extract-based system and biochemically defined system, both of which provide a great degree of freedom for bioengineering.
Up to now, the crude extract-based system is the most broadly used complex CFPS system. It processes and breaks cells to remove insoluble substances and obtain necessary biochemical components for energy generation, transcription, and translation. Theoretically, almost all species can meet the basic needs of building a cell crude extract system . A variety of CFPS systems with different cell crude extracts have been well developed . For example, products that have been commercialized include NEBExpress Cell-free E. coli Protein Synthesis System (NEB), 1-Step Human In Vitro Protein Expression Kits (Thermo Scientific), and ALiCE® Mini Kit (Sigma). According to different sources, cell extracts can be divided into prokaryotic cell extracts and eukaryotic cell extracts [12, 13]. Among them, the CFPS system of E. coli is the most widely studied CFPS system at present. Although the CFPS system based on the cell crude extract already has the advantages that the traditional cell system cannot compare, we have to admit that it still has many shortcomings. There are still instabilities and uncertainties in the cell-free system using crude cell extracts, and there are great differences between different batches . For example, a recent study found that although researchers in different laboratories use the same scheme to prepare the CFPS system, its variability is as high as 40.3% . In addition to instability, it also has component uncertainty. For example, nucleases, ribonucleases, and proteases that cannot be removed can definitely have a negative impact on translation productivity [16, 17]. To solve these problems, researchers continuously optimized the CFPS system based on crude extract and, at the same time, vigorously developed a biochemically defined system.
A typical defined CFPS system, namely, protein synthesis using recombinant elements (PURE) system, was first developed by the Shimizu group in 2001 . It is a CFPS system composed of purified components required for transcription and translation, in which all additives are completely known, and the concentration is controllable [19–21]. The PURE system has three apparent advantages over the crude extract-based system (Table 1). First, the composition of the PURE system is precise. It generally includes 36 purified proteins, tRNAs, ribosome, and necessary factors. There is no polluting protease in the PURE system, which makes the PURE system stable and deterministic. Second, the composition of each element in the PURE system can be adjusted according to different experimental needs to achieve the maximum protein expression, making the PURE system flexible [22–24]. Third, genetic code expansion or reprogramming is easier to be explored, and the manipulation of the translational machinery is easy in the PURE system [13, 25].
After nearly 20 years of research and development, the PURE system is considered to have significant advantages in basic biochemical research because of its transparent and controllable components . This paper gives a comprehensive overview of the PURE system. The basic formulation of the PURE system is first introduced. Then, how to obtain the defined ingredients and key ribosome for assembling the PURE system was demonstrated. The latest research progress in the application fields of the PURE system was discussed, including prototyping, synthesizing, and biosensing. Furthermore, this review analyzes the opportunities and challenges that the PURE system may face. It is believed that the PURE system can be combined with more disciplines and technologies in the future to better apply to the research and development in various fields.
2. Construction of PURE Systems
2.1. Primary Components
The composition of the PURE system is defined, including purified proteins, ribosome, energy, and essential factors, as shown in Figure 1. The most critical part of the PURE system is the highly purified ribosome, followed by the purified proteins. These proteins include 10 μg/mL T7 RNA polymerase for transcription, a full set of 20 tRNA synthetases for continuous tRNA aminoacylation (1900 U/mL AlaRS, 2500 U/mL ArgRS, 20 mg/mL AsnRS, 2500 U/mL AspRS, 630 U/mL CysRS, 1300 U/mL GlnRS, 1900 U/mL GluRS, 5000 U/mL GlyRS, 630 U/mL HisRS, 2500 U/mL IleRS, 3800 U/mL LeuRS, 3800 U/mL LysRS, 6300 U/mL MetRS, 1300 U/mL PheRS, 1300 U/mL ProRS, 1900 U/mL SerRS, 1300 U/mL ThrRS, 630 U/mL TrpRS, 630 U/mL TyrRS, and 3100 U/mL ValRS), translation factors for initiation, elongation, and termination (initiation factors, 2.7 μM IF1, 0.4 μM IF2, and 1.5 μM IF3; elongation factors, 0.92 μM EF-Tu, 0.66 μM EF-Ts, and 0.26 μM EF-G; release factors, 0.25 μM RF1, 0.24 μM RF2, and 0.17 μM RF3; and ribosome recycling factor, 0.5 μM RRF), and enzymes for energy cycle and regeneration (4 μg/mL creatine kinase, 3 μg/mL myokinase, 1.1 μg/mL nucleoside-diphosphate kinase, 4500 U/mL methionyl-tRNA-formyltransferase, and 2 U/mL pyrophosphatase). Maintaining the PURE system’s reactions also requires energy and other component buffers, including 2 mM ATP, 2 mM GTP, 1 mM CTP, 1 mM UTP, 20 mM creatine phosphate, 50 mM HEPES–KOH PH 7.6, 100 mM potassium glutamate, 13 mM magnesium acetate, 2 mM spermidine, 1 mM DTT, 0.3 mM 20 amino acids, 10 mg/mL 10-formyl-5,6,7,8-tetrahydrofolic acid, and 56 UA260/mL tRNAs. These purified protein components are combined with ribosomes at a concentration of 1.2 μM and the necessary templates to construct the PURE system .
In addition to homemade PURE systems, there are commercial PURE systems available, such as PUREfrex 2.0 (GeneFrontier), PURExpress (NEB) , and Magic PURE system (Creative Biolabs). Commercial PURE systems are efficient and easy to use but very expensive, around $0.6-2/microliter.
2.2. Ribosome Isolation
Ribosome is a highly complex intracellular macromolecule, mainly composed of rRNA and dozens of different r-proteins. The constituents of different species vary slightly. The ribosome of prokaryotes is composed of 65% rRNA and 35% r-protein , but the proportion of RNA and protein in eukaryotic ribosome is nearly identical . The r-protein and rRNA are organized into the ribosomal large subunit (LSU) and the ribosomal small subunit (SSU) of ribosomes. Different sedimentation coefficients permit the categorization of ribosomes into the 70S and 80S types. The 70S ribosome mainly exists in prokaryotic cells. Its SSU unit is 30S, and its LSU unit is 50S. The 80S ribosome mainly exists in eukaryotic cells. The SSU unit is 40S, and the LSU unit is 60S. During protein synthesis, the LSU and SSU of ribosomes collaborate to transform mRNA into polypeptide chains.
The ribosome as the core translational machinery is the most critical component of the PURE system, but how to purify active ribosomes is the most challenging step . The key factors for ribosome purification are cell quality and the purification strategy. The selection of strains used to purify ribosomes and the timing of cell collection impacts final ribosome quality. Compared with common strains, strains lacking RNase I, such as MRE600, A19, JE28, and Q13, are the preferred E. coli strains to purify ribosomes , reducing ribosomal RNA degradation to a certain extent. MRE600 (ATCC 29417) lacks RNase I . A19 (CGSC 5997) has six mutations of rna-19, gdhA2, his-95, relA1, spoT1, and metB1 . In JE28, hexahistidine affinity tag has been inserted at the C-terminus of the ribosomal protein L12 . Q13 is a mutant of A19 . Strains used to obtain ribosomes should grow rapidly in a rich medium at the optimum temperature and be harvested in the early and medium logarithmic stages before growth and translation begin to slow down.
The ribosome purification can be divided into two ways according to whether they have His-tag or not, as shown in Figure 2(a). One way is introducing His-tag into three ribosome subunit genes, and the ribosome can be directly and simply purified using Ni-NTA Sepharose. It has been proved that the purified ribosome labeled with His-tag in 50s or 30S ribosomal proteins has the same activity as 70S ribosome purified using traditional sucrose gradient centrifugation . After the development and improvement of this method , more laboratories can prepare purified ribosomes, because it only needs standard laboratory equipment to operate, which can further facilitate the preparation of the PURE system. The other way is traditionally purifying ribosomes by hydrophobic interaction and sucrose gradient centrifugation. Although this method can be applied to the purification of both tag-free ribosome and His-tag labeled ribosome, it requires that the laboratory is equipped to use a liquid chromatography system and ultracentrifuge . In some studies, it was found that the activity of His-tag ribosome was significantly lower than that of unlabeled variants, and in some cases, this low yield may be acceptable . Although the purification strategy of ribosomes has a significant impact on the activity of the PURE system, RNA degradation and protease pollution in the environment are inevitable in the process of ribosome purification. How to avoid such degradation and pollution is still a challenging research direction for future efforts.
2.3. Functional Proteins
Obtaining high-quality purified functional proteins except the ribosome is another key part of preparing the PURE system. After years of ongoing experimental studies, there are two main methods for purifying proteins in the PURE system. One is to purify each protein one by one (Figure 2(b)) and then add and mix each protein according to the amount required after all purification. Another purification method is combined purification from microbial consortia or bacterial artificial chromosomes. For example, a total of 30 translation factors with His-tag were encoded into three high-copy expression plasmids, and only three times of batch purification was further performed , as shown in Figure 2(c). These two methods have their own advantages and disadvantages. Although the method of individual purification takes a long time and is troublesome to operate, it can control the concentration of each protein added to the PURE system. However, while the combined purification method can greatly shorten the time required for protein purification and successfully establish the PURE system, it is unable to control the concentration of each protein. What needs to be emphasized is that, in the combined method, there are still six proteins that cannot be encoded on plasmids, which need to be purified separately, including T7 RNA polymerase, EF-Tu, myokinase, nucleoside-diphosphate kinase, pyrophosphatase, and creatine phosphate. In particular, EF-Tu needs to be purified separately, because it requires a higher level than any other translation factor.
2.4. Different Chassis
In theory, any organism can be the chassis for constructing the CFPS system, but due to still unclear translation mechanism and cumbersome preparation process, only two chassis cells are developed, including E. coli and yeast. In 2001, researchers first used E. coli to establish the PURE system, because it has clear genomic information and the characteristics of simple culture conditions, fast growth, low culture cost, simple cell lysis method, and high protein yield . Up to now, E. coli is still the most commonly used model strain of the PURE system. As people become more and more familiar with engineering the PURE system, researchers are no longer satisfied with the prokaryote research of E. coli but turn their attention to how to use eukaryotes to establish the PURE system and carry out relevant research and application.
Yeast Saccharomyces cerevisiae, as one of the most typical eukaryotes, first attracted the attention of researchers. Although cap-dependent initiation is the most complex translation stage in yeast, it requires at least 12 initiation factors and tRNA aminoacylation process, which has always been a difficulty in yeast translation. In a recent study, researchers developed CrPV IGR IRES (intergenic region internal ribosome entry site sequence from cricket paralysis virus)-containing mRNA , which enables yeast 80S ribosome to be started without promoter tRNA or any eukaryotic translation promoter. Long peptides can be synthesized in vitro by combining CrPV IGR IRES with a recombinant translation system composed of components from yeast and E. coli (yeast: elongation factors eEF1A and eEF2, tRNA mix, and recycling factor Rli1; E. coli: elongation factors eEF3, termination factors eRF1 and eRF3, and recycling factors Hbs1 and Dom34). The emergence of this yeast-derived PURE system proved that eukaryotic translation factor eIF5A could not only synthesize long peptides  but also alleviate polyproline-mediated ribosomal stall  by eIF5A and its hypusine modification.
The development of PURE systems with different chassis cells will be potentially useful for various applications. Taking the PURE system of yeast as an example, it is applicable to the design of drugs that act on eukaryotic ribosome (such as antibiotics against fungi) and the study of corresponding structures and functions. Combined with a ribosome- or mRNA-display system, this system can be used as a powerful in vitro screening system for new functional proteins, such as antibacterial peptides with cytotoxicity. It is believed that with the emergence of PURE systems developed by more different chassis cells, the PURE systems will have more application fields and can play a broader role through the combination with other systems or disciplines.
2.5. Optimization Tactics
The emergence of the PURE system has further expanded the application scope of the CFPS system , but as a protein synthesis technology, it still has the problems of high reagent cost, small reaction scale, and low yield of recombinant protein. Based on this research background, two optimization tactics have been adopted, including regulating system components and molecular crowding. Using the same reagent dosage to produce more proteins can make the PURE system relatively cheap. By adjusting the concentrations of ribosome, translation factors (especially EF-Tu), release factors, and initiation factors, the protein yield of the PURE system can be increased by up to five times. However, the concentrations of these elements are not the higher, the better. When the PURE system reaches the maximum protein output, if the concentration of each element continues to increase, the productivity of the PURE system would be inhibited [40, 48, 61–63].
In addition to adjusting the component concentrations, adding bovine serum albumin (BSA) to the PURE system or aggregating the functional proteins of the PURE system around nanoparticles can increase the environmental molecular crowding, which in turn can increase the protein synthesis in the PURE system. By cross-linking the PURE system enzymes onto quantum dots, the formed nanoaggregates can improve the synthesis of the fluorescent protein and phosphotriesterase with a 12-fold increase . This may be achieved by bringing components closer together or optimizing crowding effects that have not yet been defined.
Accordingly, adjusting the component concentrations and environmental molecular crowding can improve protein synthesis. However, it must be said that the concentrations of some elements in the PURE system cannot be adjusted even if necessary. For example, magnesium ion affects the ribosome function, but it is difficult to control its concentration because it can be chelated by negatively charged molecules, such as NTPs, creatine phosphate, and pyrophosphate. Therefore, developing more effective and convenient tactics to improve protein synthesis is still an urgent challenge that PURE systems have to face now and even in the future.
3. Cutting-Edge Applications of PURE System
The PURE system has realized the rapid expression of recombinant protein. Its flexibility, controllability, and convenient operation make it used in many application fields. This section discusses the latest research progress of the PURE system in different fields, including prototype design, incorporation of unnatural amino acids, peptide synthesis, complex protein synthesis, and biosensors.
3.1. Prototype Design
In prototyping applications, the idea is to use CFPS systems to test and optimize biosynthetic pathways before implementation and amplification in living cells. The PURE system is based on purified components and therefore is flexible and adaptable to different reaction settings. It can synthesize many proteins difficult to express in cells . As shown in Figure 3, the adaptability of the PURE system is very useful in the prototype design of candidate drugs without protein purification, which can not only synthesize various therapeutic molecules but also readily explore the effect of drugs on specific pathways . For example, one study synthesized nonribosomal peptide natural products (NRPs) indigoidine and rhabdopeptide through the PURE system . Apart from strong adaptability, the PURE system also features short response time. Shortening the time from the beginning to the end of the reaction is another important advantage of the PURE system in prototype design. Moreover, the PURE system can simulate the communication process of cells based on gene networks combined with microfluidic technology. For example, the PURE reaction generates α-haemolysin after light activation, so as to create a communication channel between individual droplets in a programmable way. The LacY transporter generated in situ by the PURE reaction realizes the active transport of signal molecules, to realize the programmed specific communication between two droplets . Prototype design is a proof-of-concept stage that must be screened and verified before industrial applications. No matter the applications of drug test or gene circuit test, the strong adaptability and short response time of the PURE system can make it the best choice of prototype design.
3.2. Unnatural Amino Acid Incorporation
The embedding of unnatural amino acids (UNAAs)  with new side chain groups can give proteins new chemical properties, forming novel protein structures and functions . It opens the door to new protein engineering, provides a new way for biological research, biotherapy, and synthetic biology, and has become a key emerging application in frontier fields . Based on orthogonal translation systems (OTSs), the UNAA incorporation methods include stop codon suppression, frameshift suppression, codon redistribution, and unnatural base pairs . The defined chemical properties of the PURE system can help to customize tRNA and aminoacyl-tRNA synthetases selectively according to application needs (Figure 4(a)). The latest research on combining OTSs with the PURE system demonstrated that selective omission of RF1 release factor into the PURE system could enhance the inhibition of biorthogonal tRNA and allow the incorporation of UNAAs at multiple sites [71, 72]. The use of mutant EF-Tu in the PURE system can improve the binding efficiency of UNAAs with a large number of side chains . Through the expansion of genetic codes  and the systematic evolution of tRNA/aminoacyl-tRNA synthetase homologs, the PURE system can incorporate more than 50 different UNAAs into proteins and can generate new chemical or functional properties in proteins by adding fluorescent markers or reaction groups [75, 76]. Twenty natural amino acids (NAAs) can also produce natural proteins with a variety of structures and functions; however, the current natural protein cannot meet the research needs of protein engineering and protein drug production. The PURE system is suitable for the application of unnatural amino acid incorporation and synthesis of complex unnatural proteins, which will make the PURE system a powerful unnatural protein engineering platform to meet the growing demand for new types of applications.
3.3. Peptide Synthesis
With the emergence and development of Flexizyme (flexible tRNA acylation ribozyme), the PURE system has become a robust platform for producing nonstandard peptides (Figure 4(b)). The combination of the PURE system and Flexizyme can produce various cyclized peptides, including backbone cyclization, backbone-side chain cyclization, and bicyclic and tricyclic peptides [77–80]. These peptides not only contain more than 300 different UNAAs, such as N-methyl and D-amino acids, but also have the potential as drugs.
Moreover, by combining the PURE system with Flexizyme tRNA aminoacylation technology and mRNA display, a powerful in vitro peptide selection platform was built [81, 82], which can generate a macrocyclic peptide library with a diversity of more than 1000. The formation of this library could contribute to the development of reagents, affinity ligands, and drugs . For example, studies have found that HIP-8 can selectively recognize active hepatocyte growth factors, which may contribute to potential cancer diagnosis and treatment . The ub4ix can specifically bind to the K48-linked ubiquitin chains and protect it from degradation by ubiquitinase. The formed macrocyclic peptide can enter cells and form a therapeutic intervention effect by inhibiting cell growth or inducing programmed death . The PURE system only contains low levels of nuclease, ribonuclease, and protease activities [86, 87], which not only allows the use of linear DNA or mRNA as the translation template of effective peptides but also has little negative impact on protein translation. These characteristics make the PURE system the first choice as the research platform for peptide synthesis.
3.4. Complex Protein
Membrane proteins play an important role in biological activities such as cell proliferation and differentiation, energy conversion, signal transduction, and material transport and account for more than 60% of the current drug target proteins . The PURE system has the characteristics of controllable reaction conditions and high toxicity tolerance, which enables it to stably synthesize complicated membrane proteins in vitro. The key support component for membrane protein synthesis is the biomembrane. Membrane simulants (such as liposomes, nanodiscs, and microsomes) can be added to the open CFPS reaction to assist the membrane protein synthesis and assembly [89–91], as shown in Figure 4(c). Using the PURE system, multiple membrane protein subunits can be prepared in parallel without cumbersome cDNA cloning or gene synthesis steps . The PURE system can also express some difficult, complex proteins that cannot be expressed in cells or traditional CFPS systems . For example, this system has been successful in producing active dihydrofolic acid reductase (DHFR) . In addition, the researchers have successfully produced insulin analogs with the same structure and affinity for receptors as those produced by yeast. This suggests that the PURE system is also suitable for expressing soluble molecules with higher-order features and multiple disulfide bridges . The PURE system can stabilize the synthesis of membrane proteins and other complex proteins, so that it could be applied to not only the basic research of biological macromolecules but also the research and development of drugs, contributing to the pharmaceutical field.
3.5. Biosensor Diagnostics
The development of the PURE system combined with freeze-drying technology or microfluidic technology can realize small volume biosensing, contributing to the application of on-site biosensors, such as point-of-care diagnosis and water quality testing , as shown in Figure 5. A new cell-free biosensor platform, RNA output sensors activated by ligand induction (ROSALIND), has been successfully developed with the PURE system. ROSALIND consists of a highly processive phage RNA polymerase, aTFs, and engineered DNA transcription templates, which together produce visible outputs upon exposure to specific ligands. Using RNA-level outputs enables the observation of signals within minutes and eliminates the requirement for sophisticated and resource-intensive protein translation to identify responses. The key advantage of ROSALIND in transcriptional RNA output is designing RNA circuits to solve the well-known shortcomings of transcription factors without protein engineering. ROSALIND can detect various compounds and elements in water, which can help fill the gap in existing water quality monitoring technology and meet the needs of communities and individuals to detect water quality quickly and cheaply .
The PURE system can be used not only for environmental monitoring but also for disease diagnosis. A low-cost colorimetric analysis method has been established, which can detect norovirus and Zika virus from clinical samples by combining cell-free RNA sensing technology with the PURE system and constant temperature amplification . The biosensor system can generate a signal proportional to the concentration of the analyte through the interaction between the sensor and the target analyte [8, 99]. The defined composition and good sensitivity of the PURE system can help develop biosensors with small volume and convenience. For example, the combination of the PURE system and colorimetric reporter enzyme can generate eye-readable biosensor readings, so that no additional equipment is required for further interpretation . This indicates that the PURE system has potential biosensing diagnostics in multiple application scenarios.
4. Conclusion and Prospects
As a powerful platform for cell-free synthetic biology technology, the PURE system developed significantly in the past five to ten years. Due to its openness, controllability, efficiency, flexibility, and many other advantages, it has been widely used, from the basic research of prototype design to the application research of biosynthesis and biosensing. However, the development of the PURE system still faces many challenges, especially in industrial applications.
Cost and scale-up are two major concerns for PURE systems in industrial manufacturing. The application expansion and development of the PURE system are limited by the cost of reagents. The high cost comes mainly from cumbersome preparation steps, low biosynthesis yield, and low system stability. Only on the basis of the cost issue being solved, the scale-up issue can be carried out further. To reduce the cost and scale up the reaction, we can seek solutions from the following aspects: simplifying the component preparation process, exploring molecular chaperones to improve protein synthesis, realizing effective energy regeneration, achieving good system stability and quality control, and designing suitable bioreactors.
As engineering problems are solved, the application potential of the PURE system needs to be further exploited. Through the fusion of artificial intelligence and computer design, natural biomolecules or networks with improved or novel functions can be prototyped and screened. With the advancement of material science, synthetic biology, physics discipline, chemistry discipline, and electronic engineering, their seamless integration with the open and flexible PURE system will open up a new world of applications in biocatalysis and human health.
|CFPS:||Cell-free protein synthesis|
|PURE:||The protein synthesis using recombinant elements|
|LSU:||Ribosomal large subunit|
|SSU:||Ribosomal small subunit|
|E. coli:||Escherichia coli|
|CrPV IGR IRES:||Intergenic region internal ribosome entry site sequence from cricket paralysis virus|
|BSA:||Bovine serum albumin|
|NRPs:||Nonribosomal peptide natural products|
|UNAAs:||Unnatural amino acids|
|OTSs:||Orthogonal translation systems|
|NAAs:||Natural amino acids|
|Flexizyme:||Flexible tRNA acylation ribozyme|
|DHFR:||Dihydrofolic acid reductase|
|ROSALIND:||RNA output sensors activated by ligand induction.|
Conflicts of Interest
The authors declare no competing interests.
Y.C. and Y.L. devised the outline of the review. Y.C. wrote the first draft. X.C. edited the manuscript. Z.W. and Y.L. revised the manuscript. Y.C. and X.C. contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (Grant No. 21878173), the National Key Research and Development Program of China (Grant No. 2018YFA0901700), and the Institute Guo Qiang, Tsinghua University (Grant No. 2021GQG1016).
- M. J. Smanski, H. Zhou, J. Claesen, B. Shen, M. A. Fischbach, and C. A. Voigt, “Synthetic biology to access and expand nature's chemical diversity,” Nature Reviews Microbiology, vol. 14, no. 3, pp. 135–149, 2016.
- G. M. Church, M. B. Elowitz, C. D. Smolke, C. A. Voigt, and R. Weiss, “Realizing the potential of synthetic biology,” Nature Reviews Molecular Cell Biology, vol. 15, no. 4, pp. 289–294, 2014.
- D. E. Cameron, C. J. Bashor, and J. J. Collins, “A brief history of synthetic biology,” Nature Reviews Microbiology, vol. 12, no. 5, pp. 381–390, 2014.
- Y. H. Wang, K. Y. Wei, and C. D. Smolke, “Synthetic biology: advancing the design of diverse genetic systems,” Annual Review of Chemical & Biomolecular Engineering, vol. 4, no. 1, pp. 69–102, 2013.
- A. Tinafar, K. Jaenes, and K. Pardee, “Synthetic biology goes cell-free,” BMC Biology, vol. 17, no. 1, p. 64, 2019.
- M. W. Nirenberg and J. H. Matthaei, “The dependence of cell-free protein synthesis inE. coliupon naturally occurring or synthetic polyribonucleotides,” Proceedings of the National Academy of Sciences, vol. 47, no. 10, pp. 1588–1602, 1961.
- J. G. Perez, J. C. Stark, and M. C. Jewett, “Cell-free synthetic biology: engineering beyond the cell,” Cold Spring Harbor Perspectives in Biology, vol. 8, no. 12, article a023853, 2016.
- D. K. Karig, “Cell-free synthetic biology for environmental sensing and remediation,” Current Opinion in Biotechnology, vol. 45, pp. 69–75, 2017.
- M. T. Smith, S. D. Berkheimer, C. J. Werner, and B. C. Bundy, “LyophilizedEscherichia coli-based cell-free systems for robust, high-density, long-term storage,” BioTechniques, vol. 56, no. 4, pp. 186–193, 2014.
- F. Katzen, G. Chang, and W. Kudlicki, “The past, present and future of cell-free protein synthesis,” Trends in Biotechnology, vol. 23, no. 3, pp. 150–156, 2005.
- B. J. L. Dopp, D. D. Tamiev, and N. F. Reuel, “Cell-free supplement mixtures: elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract,” Biotechnology Advances, vol. 37, no. 1, pp. 246–258, 2019.
- A. Zemella, L. Thoring, C. Hoffmeister, and S. Kubick, “Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems,” Chembiochem: A European journal of chemical biology, vol. 16, no. 17, pp. 2420–2431, 2015.
- R. B. Quast, D. Mrusek, C. Hoffmeister, A. Sonnabend, and S. Kubick, “Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis,” FEBS Letters, vol. 589, no. 15, pp. 1703–1712, 2015.
- M. K. Takahashi, C. A. Hayes, J. Chappell et al., “Characterizing and prototyping genetic networks with cell-free transcription- translation reactions,” Methods in Enzymology, vol. 86, pp. 60–72, 2015.
- S. D. Cole, K. Beabout, K. B. Turner et al., “Quantification of interlaboratory cell-free protein synthesis variability,” ACS Synthetic Biology, vol. 8, no. 9, pp. 2080–2091, 2019.
- D. Foshag, E. Henrich, E. Hiller et al., “The E. coli S30 lysate proteome: a prototype for cell-free protein production,” New Biotechnology, vol. 40, pp. 245–260, 2018.
- D. Garenne, C. L. Beisel, and V. Noireaux, “Characterization of the all-E. coli transcription-translation system myTXTL by mass spectrometry,” Rapid Communications in Mass Spectrometry, vol. 33, no. 11, pp. 1036–1048, 2019.
- Y. Shimizu, A. Inoue, Y. Tomari et al., “Cell-free translation reconstituted with purified components,” Nature Biotechnology, vol. 19, no. 8, pp. 751–755, 2001.
- T. Matsuura, K. Hosoda, and Y. Shimizu, “Robustness of a reconstitutedEscherichia coliprotein translation system analyzed by computational modeling,” ACS Synthetic Biology, vol. 7, no. 8, pp. 1964–1972, 2018.
- E. D. Carlson, R. Gan, C. E. Hodgman, and M. C. Jewett, “Cell-free protein synthesis: applications come of age,” Biotechnology Advances, vol. 30, no. 5, pp. 1185–1194, 2012.
- Z. Z. Sun, C. A. Hayes, J. Shin, F. Caschera, R. M. Murray, and V. Noireaux, “Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology,” Journal of Visualized Experiments, vol. 79, no. 79, article e50762, 2013.
- N. E. Gregorio, M. Z. Levine, and J. P. Oza, “A user's guide to cell-free protein synthesis,” Methods & Protocols, vol. 2, no. 1, p. 24, 2019.
- J. W. Whittaker, “Cell-free protein synthesis: the state of the art,” Biotechnology Letters, vol. 35, no. 2, pp. 143–152, 2013.
- K. Pardee, S. Slomovic, P. Q. Nguyen et al., “Portable, on-demand biomolecular manufacturing,” Cell, vol. 167, no. 1, pp. 248–259.e12, 2016.
- Z. Cui, W. A. Johnston, and K. Alexandrov, “Cell-free approach for non-canonical amino acids incorporation into polypeptides,” Frontiers in Bioengineering and Biotechnology, vol. 8, p. 1031, 2020.
- L. Grasemann, B. Lavickova, M. C. Elizondo-Cantú, and S. J. Maerkl, “OnePot PURE cell-free system,” Journal of Visualized Experiments, vol. 23, no. 172, 2021.
- R. Adiga, M. Al-Adhami, A. Andar et al., “Point-of-care production of therapeutic proteins of good-manufacturing- practice quality,” Nature Biomedical Engineering, vol. 2, no. 9, pp. 675–686, 2018.
- H. Wang, J. Li, and M. C. Jewett, “Development of a Pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements,” Synthetic Biology, vol. 3, no. 1, 2018.
- R. Kelwick, A. J. Webb, J. T. Mac Donald, and P. S. Freemont, “Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements,” Metabolic Engineering, vol. 38, pp. 370–381, 2016.
- S. J. Moore, J. T. Mac Donald, S. Wienecke et al., “Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria,” in Proceedings of the National Academy of Sciences of the United States of America, vol. 115, no. 19, pp. E4340–E4349, 2018.
- C. Guan, W. Cui, J. Cheng, L. Zhou, Z. Liu, and Z. Zhou, “Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis,” Microbial Cell Factories, vol. 15, no. 1, p. 66, 2016.
- D. E. Jeong, S. H. Park, J. G. Pan, E. J. Kim, and S. K. Choi, “Genome engineering using a synthetic gene circuit in Bacillus subtilis,” Nucleic Acids Research, vol. 43, no. 6, article e42, 2015.
- S. Guiziou, V. Sauveplane, H. J. Chang et al., “A part toolbox to tune genetic expression in Bacillus subtilis,” Nucleic Acids Research, vol. 44, no. 15, pp. 7495–7508, 2016.
- H. Sun, D. Zhao, B. Xiong, C. Zhang, and C. Bi, “Engineering Corynebacterium glutamicum for violacein hyper production,” Microbial Cell Factories, vol. 15, no. 1, p. 148, 2016.
- K. M. Smith, K. M. Cho, and J. C. Liao, “Engineering Corynebacterium glutamicum for isobutanol production,” Applied Microbiology & Biotechnology, vol. 87, no. 3, pp. 1045–1055, 2010.
- B. J. Des Soye, S. R. Davidson, M. T. Weinstock, D. G. Gibson, and M. C. Jewett, “Establishing a high-yielding cell-free protein synthesis platform derived fromVibrio natriegens,” ACS Synthetic Biology, vol. 7, no. 9, pp. 2245–2255, 2018.
- J. Li, H. Wang, Y. C. Kwon, and M. C. Jewett, “Establishing a high yielding streptomyces-based cell-free protein synthesis system,” Biotechnology and Bioengineering, vol. 114, no. 6, pp. 1343–1353, 2017.
- S. J. Moore, H. E. Lai, H. Needham, K. M. Polizzi, and P. S. Freemont, “Streptomyces venezuelae TX-TL – a next generation cell-free synthetic biology tool,” Biotechnology Journal, vol. 12, no. 4, 2017.
- D. J. Wiegand, H. H. Lee, N. Ostrov, and G. M. Church, “Establishing a cell-freeVibrio natriegensexpression system,” ACS Synthetic Biology, vol. 7, no. 10, pp. 2475–2479, 2018.
- J. Li, C. Zhang, P. Huang et al., “Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system,” Translation (Austin), vol. 5, no. 1, article e1327006, 2017.
- Z. Cui, Y. Wu, S. Mureev, and K. Alexandrov, “Oligonucleotide-mediated tRNA sequestration enables one-pot sense codon reassignment in vitro,” Nucleic Acids Research, vol. 46, no. 12, pp. 6387–6400, 2018.
- N. Laohakunakorn, L. Grasemann, B. Lavickova et al., “Bottom-up construction of complex biomolecular systems with cell-free synthetic biology,” Frontiers in Bioengineering and Biotechnology, vol. 8, p. 213, 2020.
- H. Matsubayashi and T. Ueda, “Purified cell-free systems as standard parts for synthetic biology,” Current Opinion in Chemical Biology, vol. 22, pp. 158–162, 2014.
- Y. Shimizu, T. Kanamori, and T. Ueda, “Protein synthesis by pure translation systems,” Methods, vol. 36, no. 3, pp. 299–304, 2005.
- C. Tuckey, H. Asahara, Y. Zhou, and S. Chong, “Protein synthesis using a reconstituted cell-free system,” Current Protocols in Molecular Biology, vol. 108, no. 1, p. 16.31.1-22, 2014.
- C. G. Kurland, “Molecular characterization of ribonucleic acid from Escherichia coli ribosomes: I. Isolation and molecular weights,” Journal of Molecular Biology, vol. 2, no. 2, pp. 83–91, 1960.
- D. N. Wilson and J. H. Doudna Cate, “The structure and function of the eukaryotic ribosome,” Cold Spring Harbor Perspectives in Biology, vol. 4, no. 5, 2012.
- T. Matsuura, Y. Kazuta, T. Aita, J. Adachi, and T. Yomo, “Quantifying epistatic interactions among the components constituting the protein translation system,” Molecular Systems Biology, vol. 5, no. 1, p. 297, 2009.
- M. C. Rivera, B. Maguire, and J. A. Lake, “Isolation of ribosomes and polysomes,” Cold Spring Harbor Protocols, vol. 2015, no. 3, pp. 293–299, 2015.
- C. M. Kurylo, N. Alexander, R. A. Dass et al., “Genome sequence and analysis ofEscherichia coliMRE600, a colicinogenic, nonmotile strain that lacks RNase I and the type I methyltransferase, EcoKI,” Genome Biology and Evolution, vol. 8, no. 3, pp. 742–752, 2016.
- A. M. Reiner, “Genetic locus for ribonuclease I in Escherichia coli,” Journal of Bacteriology, vol. 97, no. 3, pp. 1522-1523, 1969.
- J. Ederth, C. S. Mandava, S. Dasgupta, and S. Sanyal, “A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli,” Nucleic Acids Research, vol. 37, no. 2, article e15, 2009.
- A. M. Reiner, “Isolation and mapping of polynucleotide phosphorylase mutants of Escherichia coli,” Journal of Bacteriology, vol. 97, no. 3, pp. 1431–1436, 1969.
- H. H. Wang, P. Y. Huang, G. Xu et al., “Multiplexedin vivoHis-tagging of enzyme pathways forin vitrosingle-pot multienzyme catalysis,” ACS Synthetic Biology, vol. 1, no. 2, pp. 43–52, 2012.
- Y. Shimizu and T. Ueda, “PURE technology,” Methods in Molecular Biology, vol. 607, no. 607, pp. 11–21, 2010.
- F. Villarreal, L. E. Contreras-Llano, M. Chavez et al., “Synthetic microbial consortia enable rapid assembly of pure translation machinery,” Nature Chemical Biology, vol. 14, no. 1, pp. 29–35, 2018.
- J. Failmezger, M. Rauter, R. Nitschel, M. Kraml, and M. Siemann-Herzberg, “Cell-free protein synthesis from non-growing, stressed Escherichia coli,” Scientific Reports, vol. 7, no. 1, p. 16524, 2017.
- T. Abe, R. Nagai, H. Imataka, and N. Takeuchi-Tomita, “Reconstitution of yeast translation elongation and termination in vitro utilizing CrPV IRES-containing mRNA,” Journal of Biochemistry, vol. 167, no. 5, pp. 441–450, 2020.
- T. Abe, R. Nagai, S. Shimazaki et al., “In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide,” The Journal of Biochemistry, vol. 167, no. 5, pp. 451–462, 2020.
- R. Nagai, Y. Xu, C. Liu, A. Shimabukuro, and N. Takeuchi-Tomita, “In vitro reconstitution of yeast translation system capable of synthesizing long polypeptide and recapitulating programmed ribosome stalling,” Methods and Protocols, vol. 4, no. 3, p. 45, 2021.
- Y. Kazuta, T. Matsuura, N. Ichihashi, and T. Yomo, “Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system,” Journal of Bioscience and Bioengineering, vol. 118, no. 5, pp. 554–557, 2014.
- J. Li, L. Gu, J. Aach, and G. M. Church, “Improved cell-free RNA and protein synthesis system,” PLoS One, vol. 9, no. 9, article e106232, 2014.
- A. Doerr, E. de Reus, P. van Nies et al., “Modelling cell-free RNA and protein synthesis with minimal systems,” Physical Biology, vol. 16, no. 2, article 025001, 2019.
- M. Thakur, J. C. Breger, K. Susumu et al., “Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in PURE transcription-translation systems,” PLoS One, vol. 17, no. 3, article e0265274, 2022.
- A. S. Karim and M. C. Jewett, “A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery,” Metabolic Engineering, vol. 36, pp. 116–126, 2016.
- J. W. Bogart, M. D. Cabezas, B. Vogeli, D. A. Wong, A. S. Karim, and M. C. Jewett, “Cell-free exploration of the natural product chemical space,” Chembiochem, vol. 22, no. 1, pp. 84–91, 2021.
- E. Dubuc, P. A. Pieters, A. J. van der Linden, J. C. M. van Hest, W. T. S. Huck, and T. F. A. de Greef, “Cell-free microcompartmentalised transcription-translation for the prototyping of synthetic communication networks,” Current Opinion in Biotechnology, vol. 58, pp. 72–80, 2019.
- L. Wang, “Genetically encoding new bioreactivity,” New Biotechnology, vol. 38, pp. 16–25, 2017.
- B. J. D. Soye, J. R. Patel, F. J. Isaacs, and M. C. Jewett, “Repurposing the translation apparatus for synthetic biology,” Current Opinion in Chemical Biology, vol. 28, pp. 83–90, 2015.
- C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, and P. G. Schultz, “A general method for site-specific incorporation of unnatural amino acids into proteins,” Science, vol. 244, no. 4901, pp. 182–188, 1989.
- D. B. Johnson, J. Xu, Z. Shen et al., “RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites,” Nature Chemical Biology, vol. 7, no. 11, pp. 779–786, 2011.
- K. V. Loscha, A. J. Herlt, R. Qi, T. Huber, K. Ozawa, and G. Otting, “Multiple-site labeling of proteins with unnatural amino acids,” Angewandte Chemie International Edition, vol. 51, no. 9, pp. 2243–2246, 2012.
- Y. Doi, T. Ohtsuki, Y. Shimizu, T. Ueda, and M. Sisido, “Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system,” Journal of the American Chemical Society, vol. 129, no. 46, p. 14458-62, 2007.
- C. C. Liu and P. G. Schultz, “Adding new chemistries to the genetic code,” Annual Review of Biochemistry, vol. 79, no. 1, pp. 413–444, 2010.
- A. J. de Graaf, M. Kooijman, W. E. Hennink, and E. Mastrobattista, “Nonnatural amino acids for site-specific protein conjugation,” Bioconjugate Chemistry, vol. 20, no. 7, pp. 1281–1295, 2009.
- M. C. Hartman, K. Josephson, C. W. Lin, and J. W. Szostak, “An expanded set of amino acid analogs for the ribosomal translation of unnatural peptides,” PLoS One, vol. 2, no. 10, article e972, 2007.
- K. Ito, T. Passioura, and H. Suga, “Technologies for the synthesis of mRNA-encoding libraries and discovery of bioactive natural product-inspired non-traditional macrocyclic peptides,” Molecules, vol. 18, no. 3, pp. 3502–3528, 2013.
- T. Passioura and H. Suga, “Reprogramming the genetic code in vitro,” Trends in Biochemical Sciences, vol. 39, no. 9, pp. 400–408, 2014.
- N. K. Bashiruddin, M. Nagano, and H. Suga, “Synthesis of fused tricyclic peptides using a reprogrammed translation system and chemical modification,” Bioorganic Chemistry, vol. 61, pp. 45–50, 2015.
- Y. Yin, Q. Fei, W. Liu, Z. Li, H. Suga, and C. Wu, “Chemical and ribosomal synthesis of topologically controlled bicyclic and tricyclic peptide scaffolds primed by selenoether formation,” Angewandte Chemie. International Ed. in English, vol. 58, no. 15, pp. 4880–4885, 2019.
- T. Passioura and H. Suga, “A RaPID way to discover nonstandard macrocyclic peptide modulators of drug targets,” Chemical Communications, vol. 53, no. 12, pp. 1931–1940, 2017.
- Y. Huang, M. M. Wiedmann, and H. Suga, “RNA display methods for the discovery of bioactive macrocycles,” Chemical Reviews, vol. 119, no. 17, pp. 10360–10391, 2019.
- T. Passioura, “The road ahead for the development of macrocyclic peptide ligands,” Biochemistry, vol. 59, no. 2, pp. 139–145, 2020.
- K. Sakai, T. Passioura, H. Sato et al., “Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor,” Nature Chemical Biology, vol. 15, no. 6, pp. 598–606, 2019.
- M. Nawatha, J. M. Rogers, S. M. Bonn et al., “De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains,” Nature Chemistry, vol. 11, no. 7, pp. 644–652, 2019.
- H. Ohashi, Y. Shimizu, B. W. Ying, and T. Ueda, “Efficient protein selection based on ribosome display system with purified components,” Biochemical & Biophysical Research Communications, vol. 352, no. 1, pp. 270–276, 2007.
- T. Ueda, T. Kanamori, and H. Ohashi, “Ribosome display with the PURE technology,” Methods in Molecular Biology, vol. 607, pp. 219–225, 2010.
- M. L. Shelby, W. He, A. T. Dang, T. L. Kuhl, and M. A. Coleman, “Cell-free co-translational approaches for producing mammalian receptors: expanding the cell-free expression toolbox using nanolipoproteins,” Frontiers in Pharmacology, vol. 10, p. 744, 2019.
- F. Junge, S. Haberstock, C. Roos et al., “Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins,” New Biotechnology, vol. 28, no. 3, pp. 262–271, 2011.
- R. Sachse, S. K. Dondapati, S. F. Fenz, T. Schmidt, and S. Kubick, “Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes,” FEBS Letters, vol. 588, no. 17, pp. 2774–2781, 2014.
- J. L. Zieleniecki, Y. Nagarajan, S. Waters et al., “Cell-free synthesis of a functional membrane transporter into a tethered bilayer lipid membrane,” Langmuir, vol. 32, no. 10, pp. 2445–2449, 2016.
- R. Narumi, Y. Shimizu, M. Ukai-Tadenuma et al., “Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins,” Proceedings of the National Academy of Sciences of the United States of America, vol. 113, no. 24, pp. E3461–E3467, 2016.
- Y. J. M. Liew, Y. K. Lee, N. Khalid, N. A. Rahman, and B. C. Tan, “Cell-free expression of a plant membrane protein BrPT2 from Boesenbergia rotunda,” Molecular Biotechnology, vol. 63, no. 4, pp. 316–326, 2021.
- K. Hibi, K. Amikura, N. Sugiura et al., “Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs,” Communications Biology, vol. 3, no. 1, p. 350, 2020.
- A. B. Jensen, F. Hubalek, C. E. Stidsen et al., “Cell free protein synthesis versus yeast expression - a comparison using insulin as a model protein,” Protein Expression and Purification, vol. 186, article 105910, 2021.
- T. Tonooka, “Microfluidic device with an integrated freeze-dried cell-free protein synthesis system for small-volume biosensing,” Micromachines, vol. 12, no. 1, p. 27, 2021.
- J. K. Jung, K. K. Alam, M. S. Verosloff et al., “Cell-free biosensors for rapid detection of water contaminants,” Nature Biotechnology, vol. 38, no. 12, pp. 1451–1459, 2020.
- K. Pardee, A. A. Green, M. K. Takahashi et al., “Rapid, low-cost detection of Zika virus using programmable biomolecular components,” Cell, vol. 165, no. 5, pp. 1255–1266, 2016.
- N. Bhalla, P. Jolly, N. Formisano, and P. Estrela, “Introduction to biosensors,” Essays in Biochemistry, vol. 60, no. 1, pp. 1–8, 2016.
- C. E. Sharpes, J. B. McManus, S. M. Blum, G. E. Mgboji, and M. W. Lux, “Assessment of colorimetric reporter enzymes in the PURE system,” ACS Synthetic Biology, vol. 10, no. 11, pp. 3205–3208, 2021.
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