Nucleated synthetic cells with genetically driven intercompartment communication.

Eukaryotic cells are characterized by multiple chemically distinct compartments, one of the most notable being the nucleus. Within these compartments, there is a continuous exchange of information, chemicals, and signaling molecules, essential for coordinating and regulating cellular activities. One of the main goals of bottom-up synthetic biology is to enhance the complexity of synthetic cells by establishing functional compartmentalization. There is a need to mimic autonomous signaling between compartments, which in living cells, is often regulated at the genetic level within the nucleus. This advancement is key to unlocking the potential of synthetic cells as cell models and as microdevices in biotechnology. However, a technological bottleneck exists preventing the creation of synthetic cells with a defined nucleus-like compartment capable of genetically programmed intercompartment signaling events. Here, we present an approach for creating synthetic cells with distinct nucleus-like compartments that can encapsulate different biochemical mixtures in discrete compartments. Our system enables in situ protein expression of membrane proteins, enabling autonomous chemical communication between nuclear and cytoplasmic compartments, leading to downstream activation of enzymatic pathways within the cell.

Advanced applications of Nanodiscs-based platforms for antibodies discovery.

Due to their fundamental biological importance, membrane proteins (MPs) are attractive targets for drug discovery, with cell surface receptors, transporters, ion channels, and membrane-bound enzymes being of particular interest. However, due to numerous challenges, these proteins present underutilized opportunities for discovering biotherapeutics. Antibodies hold the promise of exquisite specificity and adaptability, making them the ideal candidates for targeting complex membrane proteins. They can target specific conformations of a particular membrane protein and can be engineered into various formats. Generating specific and effective antibodies targeting these proteins is no easy task due to several factors. The antigen's design, antibody-generation strategies, lead optimization technologies, and antibody modalities can be modified to tackle these challenges. The rational employment of cutting-edge lipid nanoparticle systems for retrieving the membrane antigen has been successfully implemented to simplify the mechanism-based therapeutic antibody discovery approach. Despite the highlighted MP production challenges, this review unequivocally underscores the advantages of targeting complex membrane proteins with antibodies and designing membrane protein antigens. Selected examples of lipid nanoparticle success have been illustrated, emphasizing the potential of therapeutic antibody discovery in this regard. With further research and development, we can overcome these challenges and unlock the full potential of therapeutic antibodies directed to target complex MPs.

Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression.

Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers' deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus's central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS's function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion.IMPORTANCEBrown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of "omics" platforms has recently uncovered the key "oxidative-hydrolytic" mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes' catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system's use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

Preparing for the future of precision medicine: synthetic cell drug regulation.

Synthetic cells are a novel class of cell-like bioreactors, offering the potential for unique advancements in synthetic biology and biomedicine. To realize the potential of those technologies, synthetic cell-based drugs need to go through the drug approval pipeline. Here, we discussed several regulatory challenges, both unique to synthetic cells, as well as challenges typical for any new biomedical technology. Overcoming those difficulties could bring transformative therapies to the market and will create a path to the development and approval of cutting-edge synthetic biology therapies. Graphical Abstract.

A Self-Regenerating Artificial Cell, that is One Step Closer to Living Cells: Challenges and Perspectives.

Controllable, self-regenerating artificial cells (SRACs) can be a vital advancement in the field of synthetic biology, which seeks to create living cells by recombining various biological molecules in the lab. This represents, more importantly, the first step on a long journey toward creating reproductive cells from rather fragmentary biochemical mimics. However, it is still a difficult task to replicate the complex processes involved in cell regeneration, such as genetic material replication and cell membrane division, in artificially created spaces. This review highlights recent advances in the field of controllable, SRACs and the strategies to achieve the goal of creating such cells. Self-regenerating cells start by replicating DNA and transferring it to a location where proteins can be synthesized. Functional but essential proteins must be synthesized for sustained energy generation and survival needs and function in the same liposomal space. Finally, self-division and repeated cycling lead to autonomous, self-regenerating cells. The pursuit of controllable, SRACs will enable authors to make bold advances in understanding life at the cellular level, ultimately providing an opportunity to use this knowledge to understand the nature of life.

Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System.

Lysate-based cell-free expression (CFE) systems are accessible platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs). NRPSs are large (>100 kDa), modular enzyme complexes that synthesize bioactive peptide natural products. This synthetic process is analogous to transcription/translation (TX/TL) in lysates, resulting in potential resource competition between NRPS expression and NRPS activity in cell-free environments. Moreover, CFE conditions depend on the size and structure of the protein. Here, a reporter system for rapidly investigating and optimizing reaction environments for NRPS CFE is described. This strategy is demonstrated in E. coli lysate reactions using blue pigment synthetase A (BpsA), a model NRPS, carrying a C-terminal tetracysteine (TC) tag which forms a fluorescent complex with the biarsenical dye, FlAsH. A colorimetric assay was adapted for lysate reactions to detect the blue pigment product, indigoidine, of cell-free expressed BpsA-TC, confirming that the tagged enzyme is catalytically active. An optimized protocol for end point TC/FlAsH complex measurements in reactions enables quick comparisons of full-length BpsA-TC expressed under different reaction conditions, defining unique requirements for NRPS expression that are related to the protein's catalytic activity and size. Importantly, these protein-dependent CFE conditions enable higher indigoidine titer and improve the expression of other monomodular NRPSs. Notably, these conditions differ from those used for the expression of superfolder GFP (sfGFP), a common reporter for optimizing lysate-based CFE systems, indicating the necessity for tailored reporters to optimize expression for specific enzyme classes. The reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.

T7Max transcription system.

BACKGROUND: Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. RESULTS: Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. CONCLUSIONS: The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

A Facile Method to Coat Nanoparticles with Lipid Bilayer Membrane: Hybrid Silica Nanoparticles Disguised as Biomembrane Vesicles by Particle Penetration of Concentrated Lipid Layers.

Natural membrane vesicles, including extracellular vesicles and enveloped viruses, participate in various events in vivo. To study and manipulate these events, biomembrane-coated nanoparticles inspired by natural membrane vesicles are developed. Herein, an efficient method is presented to prepare organic-inorganic hybrid materials in high yields that can accommodate various lipid compositions and particle sizes. To demonstrate this method, silica nanoparticles are passed through concentrated lipid layers prepared using density gradient centrifugation, followed by purification, to obtain lipid membrane-coated nanoparticles. Various lipids, including neutral, anionic, and cationic lipids, are used to prepare concentrated lipid layers. Single-particle analysis by imaging flow cytometry determines that silica nanoparticles are uniformly coated with a single lipid bilayer. Moreover, cellular uptake of silica nanoparticles is enhanced when covered with a lipid membrane containing cationic lipids. Finally, cell-free protein expression is applied to embed a membrane protein, namely the Spike protein of severe acute respiratory syndrome coronavirus 2, into the coating of the nanoparticles, with the correct orientation. Therefore, this method can be used to develop organic-inorganic hybrid nanomaterials with an inorganic core and a virus-like coating, serving as carriers for targeted delivery of cargos such as proteins, DNA, and drugs.

Cell-Free Gene Expression from DNA Brushes.

Linear double-stranded DNA polymers coding for synthetic genes immobilized on a surface form a brush as a center for cell-free gene expression, with DNA density 102-103 fold higher than in bulk solution reactions. A brush localizes the transcription-translation machinery in cell extracts or in cell-free reconstituted reactions from purified components, creating a concentrated source of RNA and proteins. Newly synthesized molecules can form circuits regulating gene expression in the same brush or adjacent ones. They can also assemble into functional complexes and machines such as ribosomal units, then analyzed by capture on prepatterned antibodies or by cascaded reactions. DNA brushes are arranged as a single center or multiple ones on a glass coverslip, in miniaturized compartments carved in silicon wafers, or in elastomeric microfluidic devices. Brushes create genetically programmable artificial cells with steady-state dynamics of protein synthesis. Here, we provide the basic procedure for surface patterning, DNA immobilization, capture of protein products on antibody traps and fluorescent imaging. The method of DNA brush surface patterning enables simple parallelization of cell-free gene expression reactions for high throughput studies with increased imaging sensitivity.

Applications of Cell-Free Synthesized Membrane Protein Precipitates.

Cell-free protein expression systems are new core platforms for membrane protein synthesis. Expression in the presence of supplied artificial hydrophobic environments such as nanomembranes or micelles allows the co-translational solubilization and folding of membrane proteins. In the absence of hydrophobic compounds, the synthesized membrane proteins quantitatively precipitate, while frequently still retaining a significant part of folded structural elements. This so-called precipitate-forming cell-free (P-CF) expression mode is a very effective and reliable approach for numerous applications. Even from complex membrane proteins such as G-protein coupled receptors or large transporters, significant amounts of such precipitates can be synthesized within few hours. The precipitates can be solubilized in detergents or reconstituted into membranes for subsequent structural or functional analysis. Harsh denaturation and refolding procedures as known from the treatment of bacterial inclusion bodies are usually not required.This strategy is particularly interesting for applications requiring large amounts of membrane protein or fast access to a sample. It is further an excellent tool for the production of membrane protein antigens suitable for antibody generation. The purification of the precipitates in downstream processing is streamlined as only few proteins from the cell-free lysate may co-precipitate with the synthesized membrane protein. For most applications, a one-step affinity chromatography by taking advantage of small purification tags attached to the membrane protein target is sufficient. We give an overview on current applications of P-CF precipitates and describe the underlying techniques in detail. We furthermore provide protocols for the successful crystallization and NMR analysis of P-CF synthesized membrane proteins exemplified with the diacylglycerol kinase (DAGK). In addition, we describe the functional characterization of a P-CF synthesized large eukaryotic transporter.

Leishmania tarentolae cell-free based approach for rapid anitbody-antigen interaction analysis.

Rapid techniques for producing high-quality recombinant proteins are essential for fast protein functional analysis, as well as various screening applications. Cell-free protein expression is an enabling tool in protein research capable of producing high-quality proteins within a few hours. In this chapter, we describe the use of a Leishmania tarentolae-based cell-free expression system to produce antibody fragments coupled to the analysis of their interaction with their ligands. Interaction analysis is performed using the scalable and sensitive AlphaLISA bead proximity assay. The method presented in this chapter offers a rapid and inexpensive approach for production of putative interacting protein pairs, as well as a multiplexable approach for their rapid interaction analysis.

Toward synthetic life: Biomimetic synthetic cell communication.

Engineering synthetic minimal cells provide a controllable chassis for studying the biochemical principles of natural life, increasing our understanding of complex biological processes. Recently, synthetic cell engineering has enabled communication between both natural live cells and other synthetic cells. A system such as these enable studying interactions between populations of cells, both natural and artificial, and engineering small molecule cell communication protocols for a variety of basic research and practical applications. In this review, we summarize recent progress in engineering communication between synthetic and natural cells, and we speculate about the possible future directions of this work.

Controlled metabolic cascades for protein synthesis in an artificial cell.

In recent years, researchers have been pursuing a method to design and to construct life forms from scratch - in other words, to create artificial cells. In many studies, artificial cellular membranes have been successfully fabricated, allowing the research field to grow by leaps and bounds. Moreover, in addition to lipid bilayer membranes, proteins are essential factors required to construct any cellular metabolic reaction; for that reason, different cell-free expression systems under various conditions to achieve the goal of controlling the synthetic cascades of proteins in a confined area have been reported. Thus, in this review, we will discuss recent issues and strategies, enabling to control protein synthesis cascades that are being used, particularly in research on artificial cells.

Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics.

Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols and optimized reaction conditions have been established for the production of cell-free gene expression systems. One of the crucial steps during the preparation of cell extract-based expression systems is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here we evaluate the utility of an E. coli cell extract, which was prepared using a combination of lysozyme incubation and a gentle sonication step. As quality measure, we demonstrate the cell-free expression of YFP at concentrations up to 0.6 mg/mL. In addition, we produced and assembled T7 bacteriophages up to a titer of 108  PFU/mL. State-of-the-art quantitative proteomics was used to compare the produced extracts with each other and with a commercial extract. The differences in protein composition were surprisingly small between lysozyme-assisted sonication (LAS) extracts, but we observed an increase in the release of DNA-binding proteins for increasing numbers of sonication cycles. Proteins taking part in carbohydrate metabolism, glycolysis, amino acid and nucleotide related pathways were found to be more abundant in the LAS extract, while proteins related to RNA modification and processing, DNA modification and replication, transcription regulation, initiation, termination and the TCA cycle were found enriched in the commercial extract.

High-Efficiency Protection of Linear DNA in Cell-Free Extracts from Escherichia coli and Vibrio natriegens.

The field of cell-free synthetic biology is an emerging branch of engineered biology that allows for rapid prototyping of biological designs and, in its own right, is becoming a venue for the in vitro operation of gene circuit-based sensors and biomanufacturing. To date, the related DNA encoded tools that operate in cell-free reactions have primarily relied on plasmid DNA inputs, as linear templates are highly susceptible to degradation by exonucleases present in cell-free extracts. This incompatibility has precluded significant throughput, time and cost benefits that could be gained with the use of linear DNA in the cell-free expression workflow. Here to tackle this limitation, we report that terminal incorporation of Ter binding sites for the DNA-binding protein Tus enables highly efficient protection of linear expression templates encoding mCherry and deGFP. In Escherichia coli extracts, our method compares favorably with the previously reported GamS-mediated protection scheme. Importantly, we extend the Tus-Ter system to Vibrio natriegens extracts, and demonstrate that this simple and easily implemented method can enable an unprecedented plasmid-level expression from linear templates in this emerging chassis organism.

Screening Methods for Cell-Free Synthesized GPCR/Nanoparticle Samples.

Cell-free protein expression systems and lipid nanoparticle technologies are core platforms for membrane protein synthesis. The implementation of preassembled nanodiscs allows the co-translational insertion of membrane proteins into tailored lipid bilayers in the absence of any artificial hydrophobic compounds. This strategy is particularly interesting for detergent sensitive or otherwise critical membrane proteins such as G-protein-coupled receptors (GPCRs). Cell-free expression reactions are completed within a day and the formed GPCR/nanodisc particles can be purified directly out of the reaction mixture by affinity tags and without any further manipulation. The streamlined procedure reduces risk of GPCR denaturation and the sample quality can further be supported by supplying chaperones or other beneficial compounds directly into the expression reactions.GPCRs inserted into nanoparticle membranes are excellent tools for a variety of applications such as ligand screening, engineering or even structural characterization. In this chapter, we provide protocols for the reaction set-up and efficient cell-free production of functionally folded GPCRs reaching µM concentrations in the final expression reactions. We further exemplify the tuning of GPCR sample quality and discuss their application for throughput ligand screening and for the analysis of ligand-binding characteristics.

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology.

Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.

Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems.

Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13 C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of "local" deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39.

Reconstituting Natural Cell Elements in Synthetic Cells.

Building a live cell from non-living building blocks would be a fundamental breakthrough in biological sciences, and it would enable engineering new lineages of life, not directly descendant of the Last Universal Common Ancestor. Fully engineered synthetic cells will have architectures that can be radically different from the natural cells, yet most life processes reconstituted in synthetic cells so far are built from natural and biosimilar building blocks. Most natural processes have already been reconstituted in synthetic cell chassis. This paper summarizes recent advancements in using non-living building blocks to reconstitute some of the most crucial features of living systems in a fully engineerable chassis of a synthetic cell.