Cell-free protein synthesis with technical additives - expanding the parameter space of in vitro gene expression.

Biocatalysis has established itself as a successful tool in organic synthesis. A particularly fast technique for screening enzymes is the in vitro expression or cell-free protein synthesis (CFPS). The system is based on the transcription and translation machinery of an extract-donating organism to which substrates such as nucleotides and amino acids, as well as energy molecules, salts, buffer, etc., are added. After successful protein synthesis, further substrates can be added for an enzyme activity assay. Although mimicking of cell-like conditions is an approach for optimization, the physical and chemical properties of CFPS are not well described yet. To date, standard conditions have mainly been used for CFPS, with little systematic testing of whether conditions closer to intracellular conditions in terms of viscosity, macromolecules, inorganic ions, osmolarity, or water content are advantageous. Also, very few non-physiological conditions have been tested to date that would expand the parameter space in which CFPS can be performed. In this study, the properties of an Escherichia coli extract-based CFPS system are evaluated, and the parameter space is extended to high viscosities, concentrations of inorganic ion and osmolarity using ten different technical additives including organic solvents, polymers, and salts. It is shown that the synthesis of two model proteins, namely superfolder GFP (sfGFP) and the enzyme truncated human cyclic GMP-AMP synthase fused to sfGFP (thscGAS-sfGFP), is very robust against most of the tested additives.

Adeno-Associated Virus 5 Protein Particles Produced by E. coli Cell-Free Protein Synthesis.

Recombinant adeno-associated viruses (rAAVs) have emerged as important tools for gene therapy and, more recently, vaccine development. Nonetheless, manufacturing can be costly and time-consuming, emphasizing the importance of alternative production platforms. We investigate the potential of E. coli-based cell-free protein synthesis (CFPS) to produce recombinant AAV5 virus-like particles (VLPs). AAV5 virus protein 3 (VP3) constructs, both with and without Strep-tag II, were expressed with CFPS. Lower reaction temperatures resulted in increased solubility, with the untagged variant containing nearly 90% more soluble VLP VP3 protein at 18 °C than at 37 °C. Affinity chromatography of N-terminally Strep(II)-tagged VP3 enabled successful isolation with minimal processing. DLS and TEM confirmed the presence of ∼20 nm particles. Furthermore, the N-terminally tagged AAV5 VP3 VLPs were biologically active, successfully internalizing into HeLa cells. This study describes an innovative approach to AAV VLP production using E. coli-based CFPS, demonstrating its potential for rapid and biologically active AAV VLP synthesis.

Recent progress in one-pot enzymatic synthesis and regeneration of high-value cofactors.

One-pot enzymatic synthesis is flourishing in synthetic chemistry, heralding a sustainable and green era. Recent advancements enable the creation of complex enzymatic prosthetic groups and regeneration of enzymatic cofactors such as S-adenosylmethionine. The next frontier is to develop the effective and innovative cofactors for essential micronutrients, metabolic modulators, and biomedicines.

Applications of cell free protein synthesis in protein design.

In protein design, the ultimate test of success is that the designs function as desired. Here, we discuss the utility of cell free protein synthesis (CFPS) as a rapid, convenient and versatile method to screen for activity. We champion the use of CFPS in screening potential designs. Compared to in vivo protein screening, a wider range of different activities can be evaluated using CFPS, and the scale on which it can easily be used-screening tens to hundreds of designed proteins-is ideally suited to current needs. Protein design using physics-based strategies tended to have a relatively low success rate, compared with current machine-learning based methods. Screening steps (such as yeast display) were often used to identify proteins that displayed the desired activity from many designs that were highly ranked computationally. We also describe how CFPS is well-suited to identify the reasons designs fail, which may include problems with transcription, translation, and solubility, in addition to not achieving the desired structure and function.

Bioengineering lipid-based synthetic cells for therapeutic protein delivery.

Synthetic cells (SCs) offer a promising approach for therapeutic protein delivery, combining principles from synthetic biology and drug delivery. Engineered to mimic natural cells, SCs provide biocompatibility and versatility, with precise control over their architecture and composition. Protein production is essential in living cells, and SCs aim to replicate this process using compartmentalized cell-free protein synthesis systems within lipid bilayers. Lipid bilayers serve as favored membranes in SC design due to their similarity to the biological cell membrane. Moreover, engineering lipidic membranes enable tissue-specific targeting and immune evasion, while stimulus-responsive SCs allow for triggered protein production and release. This Review explores lipid-based SCs as platforms for therapeutic protein delivery, discussing their design principles, functional attributes, and translational challenges and potential.

Cell-Free Protein Expression by a Reconstituted Transcription-Translation System Energized by Sugar Catabolism.

Cooperation between catabolism and anabolism is crucial for maintaining homeostasis in living cells. The most fundamental systems for catabolism and anabolism are the glycolysis of sugars and the transcription-translation (TX-TL) of DNA, respectively. Despite their importance in living cells, the in vitro reconstitution of their cooperation through purified factors has not been achieved, which hinders the elucidation of the design principle in living cells. Here, we reconstituted glycolysis using sugars and integrated it with the PURE system, a commercial in vitro TX-TL kit composed of purified factors. By optimizing key parameters, such as glucokinase and initial phosphate concentrations, we determined suitable conditions for their cooperation. The optimized system showed protein synthesis at up to 33% of that of the original PURE system. We observed that ATP consumption in upstream glycolysis inhibits TX-TL and that this inhibition can be alleviated by the co-addition of glycolytic intermediates, such as glyceraldehyde 3-phosphate, with glucose. Moreover, the system developed here simultaneously synthesizes a subset of its own enzymes, that is, glycolytic enzymes, in a single test tube, which is a necessary step toward self-replication. As glycolysis and TX-TL provide building blocks for constructing cells, the integrated system can be a fundamental material for reconstituting living cells from purified factors.

Cell-Free Synthesis of Bunyavirales Proteins in View of Their Structural Characterization by Nuclear Magnetic Resonance.

The Rift Valley fever virus is one of the bunyaviruses on the WHO's priority list of pathogens that may cause future pandemics. A better understanding of disease progression and viral pathogenesis is urgently needed to develop treatments. The non-structural proteins NSs and NSm of human pathogenic bunyaviruses represent promising therapeutic targets, as they are often key virulence factors. However, their function is still poorly understood, and their structure is yet unknown, mainly because no successful production of these highly complex proteins has been reported. Here we propose a powerful combination of wheat germ cell-free protein synthesis and NMR to study the structure of these proteins and in particular detail cell-free synthesis and lipid reconstitution methods that can be applied to complex membrane proteins.

Emerging Designs and Applications for Biomembrane Biosensors.

Nature has inspired the development of biomimetic membrane sensors in which the functionalities of biological molecules, such as proteins and lipids, are harnessed for sensing applications. This review provides an overview of the recent developments for biomembrane sensors compatible with either bulk or planar sensing applications, namely using lipid vesicles or supported lipid bilayers, respectively. We first describe the individual components required for these sensing platforms and the design principles that are considered when constructing them, and we segue into recent applications being implemented across multiple fields. Our goal for this review is to illustrate the versatility of nature's biomembrane toolbox and simultaneously highlight how biosensor platforms can be enhanced by harnessing it.

Bacterial glycoengineering: Cell-based and cell-free routes for producing biopharmaceuticals with customized glycosylation.

Glycosylation plays a pivotal role in tuning the folding and function of proteins. Because most human therapeutic proteins are glycosylated, understanding and controlling glycosylation is important for the design, optimization, and manufacture of biopharmaceuticals. Unfortunately, natural eukaryotic glycosylation pathways are complex and often produce heterogeneous glycan patterns, making the production of glycoproteins with chemically precise and homogeneous glycan structures difficult. To overcome these limitations, bacterial glycoengineering has emerged as a simple, cost-effective, and scalable approach to produce designer glycoprotein therapeutics and vaccines in which the glycan structures are engineered to reduce heterogeneity and improve biological and biophysical attributes of the protein. Here, we discuss recent advances in bacterial cell-based and cell-free glycoengineering that have enabled the production of biopharmaceutical glycoproteins with customized glycan structures.

Split T7 switch-mediated cell-free protein synthesis system for detecting target nucleic acids.

Cell-free protein synthesis (CFPS) reactions can be used to detect nucleic acids. However, most CFPS systems rely on a toehold switch and exhibit the following critical limitations: (i) off-target signals due to leaky translation in the absence of target nucleic acids, (ii) a suboptimal detection limit of approximately 30 nM without pre-amplification, and (iii) labor-intensive screening processes due to sequence constraints for the target nucleic acids. To overcome these shortcomings, we developed a new split T7 switch-mediated CFPS system in which the split T7 promoter was applied to a three-way junction structure to selectively initiate transcription-translation only in the presence of target nucleic acids. Both fluorescence and colorimetric detection systems were constructed by employing different reporter proteins. Notably, we introduced the self-complementation of split fluorescent proteins to streamline preparation of the proposed system, enabling versatile applications. Operation of this one-pot approach under isothermal conditions enabled the detection of target nucleic acids at concentrations as low as 10 pM, representing more than a thousand times improvement over previous toehold switch-based approaches. Furthermore, the proposed system demonstrated high specificity in detecting target nucleic acids and compatibility with various reporter proteins encoded in the expression region. By eliminating issues associated with the previous toehold switch system, our split T7 switch-mediated CFPS system could become a core platform for detecting various target nucleic acids.

Evaluation in mice of cell-free produced CT584 as a Chlamydia vaccine antigen.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs' weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production.

N-Terminal Amino Acid Affects the Translation Efficiency at Lower Temperatures in a Reconstituted Protein Synthesis System.

The Escherichia coli (E. coli)-based protein synthesis using recombinant elements (PURE) system is a cell-free protein synthesis system reconstituted from purified factors essential for E. coli translation. The PURE system is widely used for basic and synthetic biology applications. One of the major challenges associated with the PURE system is that the protein yield of the system varies depending on the protein. Studies have reported that the efficiency of translation is significantly affected by nucleotide and amino acid sequences, especially in the N-terminal region. Here, we investigated the inherent effect of various N-terminal sequences on protein synthesis using the PURE system. We found that a single amino acid substitution in the N-terminal region significantly altered translation efficiency in the PURE system, especially at low temperatures. This result gives us useful suggestions for the expression of the protein of interest in vitro and in vivo.

Engineering the Signal Resolution of a Paper-Based Cell-Free Glutamine Biosensor with Genetic Engineering, Metabolic Engineering, and Process Optimization.

Specialized cancer treatments have the potential to exploit glutamine dependence to increase patient survival rates. Glutamine diagnostics capable of tracking a patient's response to treatment would enable a personalized treatment dosage to optimize the tradeoff between treatment success and dangerous side effects. Current clinical glutamine testing requires sophisticated and expensive lab-based tests, which are not broadly available on a frequent, individualized basis. To address the need for a low-cost, portable glutamine diagnostic, this work engineers a cell-free glutamine biosensor to overcome assay background and signal-to-noise limitations evident in previously reported studies. The findings from this work culminate in the development of a shelf-stable, paper-based, colorimetric glutamine test with a high signal strength and a high signal-to-background ratio for dramatically improved signal resolution. While the engineered glutamine test is important progress towards improving the management of cancer and other health conditions, this work also expands the assay development field of the promising cell-free biosensing platform, which can facilitate the low-cost detection of a broad variety of target molecules with high clinical value.

Transporter function characterization via continuous-exchange cell-free synthesis and solid supported membrane-based electrophysiology.

Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as KM, IMAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.

Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution.

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α.

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed.

A highly optimized human in vitro translation system.

In vitro translation is an important method for studying fundamental aspects of co- and post-translational gene regulation, as well as for protein expression in the laboratory and on an industrial scale. Here, by re-examining and improving a human in vitro translation system (HITS), we were able to develop a minimal system where only four components are needed to supplement human cell lysates. Functional characterization of our improved HITS revealed the synergistic effect of mRNA capping and polyadenylation. Furthermore, we found that mRNAs are translated with an efficiency equal to or higher than existing state-of-the-art mammalian in vitro translation systems. Lastly, we present an easy preparation procedure for cytoplasmic extracts from cultured HeLa cells, which can be performed in any cell culture laboratory. These methodological advances will allow HITSs to become a widespread tool in basic molecular biology research.

Validation of Cell-Free Protein Synthesis Aboard the International Space Station.

Cell-free protein synthesis (CFPS) is a rapidly maturing in vitro gene expression platform that can be used to transcribe and translate nucleic acids at the point of need, enabling on-demand synthesis of peptide-based vaccines and biotherapeutics as well as the development of diagnostic tests for environmental contaminants and infectious agents. Unlike traditional cell-based systems, CFPS platforms do not require the maintenance of living cells and can be deployed with minimal equipment; therefore, they hold promise for applications in low-resource contexts, including spaceflight. Here, we evaluate the performance of the cell-free platform BioBits aboard the International Space Station by expressing RNA-based aptamers and fluorescent proteins that can serve as biological indicators. We validate two classes of biological sensors that detect either the small-molecule DFHBI or a specific RNA sequence. Upon detection of their respective analytes, both biological sensors produce fluorescent readouts that are visually confirmed using a hand-held fluorescence viewer and imaged for quantitative analysis. Our findings provide insights into the kinetics of cell-free transcription and translation in a microgravity environment and reveal that both biosensors perform robustly in space. Our findings lay the groundwork for portable, low-cost applications ranging from point-of-care health monitoring to on-demand detection of environmental hazards in low-resource communities both on Earth and beyond.

Cell-Free Protein Expression in Polymer Materials.

While synthetic biology has advanced complex capabilities such as sensing and molecular synthesis in aqueous solutions, important applications may also be pursued for biological systems in solid materials. Harsh processing conditions used to produce many synthetic materials such as plastics make the incorporation of biological functionality challenging. One technology that shows promise in circumventing these issues is cell-free protein synthesis (CFPS), where core cellular functionality is reconstituted outside the cell. CFPS enables genetic functions to be implemented without the complications of membrane transport or concerns over the cellular viability or release of genetically modified organisms. Here, we demonstrate that dried CFPS reactions have remarkable tolerance to heat and organic solvent exposure during the casting processes for polymer materials. We demonstrate the utility of this observation by creating plastics that have spatially patterned genetic functionality, produce antimicrobials in situ, and perform sensing reactions. The resulting materials unlock the potential to deliver DNA-programmable biofunctionality in a ubiquitous class of synthetic materials.

Using High-Throughput Experiments To Screen N-Glycosyltransferases with Altered Specificities.

The important roles that protein glycosylation plays in modulating the activities and efficacies of protein therapeutics have motivated the development of synthetic glycosylation systems in living bacteria and in vitro. A key challenge is the lack of glycosyltransferases that can efficiently and site-specifically glycosylate desired target proteins without the need to alter primary amino acid sequences at the acceptor site. Here, we report an efficient and systematic method to screen a library of glycosyltransferases capable of modifying comprehensive sets of acceptor peptide sequences in parallel. This approach is enabled by cell-free protein synthesis and mass spectrometry of self-assembled monolayers and is used to engineer a recently discovered prokaryotic N-glycosyltransferase (NGT). We screened 26 pools of site-saturated NGT libraries to identify relevant residues that determine polypeptide specificity and then characterized 122 NGT mutants, using 1052 unique peptides and 52,894 unique reaction conditions. We define a panel of 14 NGTs that can modify 93% of all sequences within the canonical X-1-N-X+1-S/T eukaryotic glycosylation sequences as well as another panel for many noncanonical sequences (with 10 of 17 non-S/T amino acids at the X+2 position). We then successfully applied our panel of NGTs to increase the efficiency of glycosylation for three protein therapeutics. Our work promises to significantly expand the substrates amenable to in vitro and bacterial glycoengineering.