Selective Elimination of Mast Cells via Siglec-6-Targeted Nanodelivery of Drug Payload.

Cell-free protein synthesis (CFPS) has the potential to reduce the cost of biologics manufacturing through simplified lysates, high-throughput workflows, and streamlined downstream purification; however, high reagent costs and inconsistent product yields remain significant barriers to industrial adoption. A key limitation is the lack of cost-effective, high-throughput methods to monitor metabolite dynamics during the CFPS optimization. To address this challenge, we developed a fluorescent assay that enables the real-time monitoring of transcription, translation, and key metabolite pools. The fluorescent assay was used to identify and address technical challenges and produce insights that were subsequently used to engineer a minimal CFPS platform that costs nearly 97.5% less than commercial systems but still attains comparable protein yields. More broadly, our work introduces a generalizable framework for interrogating and engineering CFPS systems through the real-time observation of transcription and metabolism.

In vitro directed evolution in synthetic microcompartments can support the evolution of genes with functions beyond affinity. The main challenge in implementing this strategy is the need to incorporate no more than a single DNA template molecule per microcompartment, thereby establishing a robust genotype-phenotype linkage, but that results in slow, inconsistent in vitro transcription and translation (IVTT) and poor DNA recovery after selection or screening. To address this challenge, we previously developed CADGE (Clonal Amplification-enhanceD Gene Expression), a strategy implemented in PURE (Protein synthesis Using Recombinant Elements) systems that allows the clonal amplification of linear gene-encoding DNA and coupled, in situ transcription-translation of the gene of interest. However, we found that current commercial PURE systems support substantially lower DNA replication than previously reported. Here, we restore DNA amplification in CADGE to previously reported levels by replacing vendor-supplied energy mixes with homemade counterparts previously shown to enhance rolling circle amplification.

Protein synthesis in cell-free protein synthesis systems often exhibits nonintuitive input-output relationships. In the PURE system, a reconstituted cell-free system, protein production peaked at low elongation factor Tu (EF-Tu) concentrations and decreased at higher concentrations, resulting in a characteristic bell-shaped profile. Here, we investigated the origin of this behavior using a detailed mechanistic model of translation in the PURE system, designated as ePURE, which describes the reaction dynamics of hundreds of molecular species and reactions. Our computational analysis suggested that excess EF-Tu sequesters the initiator tRNA (tRNAfMet) into nonproductive EF-Tu·GTP·Met-tRNAfMet complexes, thereby depleting the pool of initiator tRNA available for translation initiation. This suppression arises from competition for a limited molecular resource rather than from direct inhibition. Based on this mechanism, we predicted that increasing the concentrations of tRNAfMet and methionyl-tRNA formyltransferase would eliminate the bell-shaped dependence, and experimentally confirmed this prediction. Under these modified conditions, the bell-shaped response disappeared and protein production was enhanced. These findings demonstrate how mechanistic computational models can reveal hidden constraints underlying nonintuitive input-output relationships in complex biochemical networks and guide the rational optimization of cell-free protein synthesis systems.

Energy-supplying molecules are essential for biological processes, particularly for transcription and translation. Cell-free protein synthesis (CFPS) systems are powerful tools for in vitro protein production, offering flexibility for applications ranging from high-throughput protein screening to therapeutic protein production. However, energy regeneration in CFPS remains a key challenge, particularly for large-scale or resource-constrained settings. In this study, we introduce phosphoserine (PS) as a simple, cost-effective alternative secondary energy source, capable of partially or fully replacing 3-phosphoglycerate (PGA), the commonly used energy donor in E. coli lysate-based CFPS, whose availability is often limited. By supplementing CFPS reactions with PS, we demonstrate significant improvements in protein yield and cost-efficiency, achieving a 2-fold increase in protein production. Importantly, PS enhancement is maintained across lysate batches and protein targets. Furthermore, we offer affordable CFPS compositions that retain protein synthesis, making the system more accessible for resource-limited settings. Additionally, we show that higher PS concentrations, while reducing final protein yield, extend the reaction duration by more than 2-fold. Therefore, the incorporation of PS as an alternative energy donor enables a tunable modality for balancing protein yield, reaction longevity, and cost. Our data support a model in which PS enhances CFPS via the serine biosynthesis pathway by modulating flux between serine production and glycolysis to support ATP regeneration. Lastly, we validate the use of these optimized CFPS compositions within synthetic cells (SCs). This study establishes PS as a promising energy source for E. coli lysate-based CFPS systems, paving the way for enhanced and economical protein synthesis platforms tailored to diverse clinical, biotechnological, and industrial needs.

The structural analysis of the ecto- or cytosolic domains of membrane proteins is frequently realized through the analysis of isolated domains, i.e., by using a divide-and-conquer approach, as membrane protein expression remains a laborious task. However, the membrane environment can influence the structural features of the protein. Here, we investigated the membrane-bound form of the cytosolic zinc finger domain of the Crimean-Congo hemorrhagic fever virus glycoprotein n (GnTMcyto), which had previously been studied in isolation. We obtained the membrane-reconstituted protein by cell-free protein synthesis and analyzed it using 1H-detected solid-state NMR. We show that the overall structure of the zinc finger motif is conserved in GnTMcyto, but interestingly observe that the membrane-bound version has two major conformations, as revealed by peak doubling. This likely indicates that the cytosolic domain forms a multimer in its membrane-bound form, with an at least partially asymmetric conformation. NMR relaxation measurements further reveal that the protein includes dynamic components, providing a possible rationale for the absence of Gncyto in cryo-EM structures of the envelope glycoproteins in other Bunyaviricetes.

Wheat germ extract (WGE)-based cell‑free translation is a robust and versatile platform for small‑scale, high‑throughput production of diverse eukaryotic proteins. Although the system has been optimised for efficient protein synthesis using cap‑independent translation‑compatible vectors and bilayer in vitro translation formats, it has traditionally relied on separate transcription and translation steps. To streamline this workflow, a bilayer, coupled in vitro transcription-translation (cIVTT) protocol was developed in which both processes are integrated into a single reaction. After optimisation of the reaction mixture composition and reaction conditions, protein yields comparable to those obtained with the conventional two‑step bilayer in vitro translation method were achieved. Notably, the cIVTT protocol remained effective at reduced temperatures, thereby supporting the enhanced production of human cardiac muscle Troponin I (cTnI), a protein with demanding folding requirements. This simplified and time‑efficient approach enables microgram‑scale protein production and is well suited for high‑throughput applications. KEY POINTS: • Streamlined WGE cell-free protein synthesis workflow • No plasmid linearisation needed before coupled in vitro transcription/translation • Soluble eukaryotic protein yields on par with two‑step bilayer format.

We demonstrate the use of fluorescence correlation spectroscopy (FCS) to characterize fluorescently-labeled protein production. We use cell-free protein synthesis to express the protein YFP-CopB, a fusion of Chlamydia Outer Protein (Cop) B and Yellow Fluorescent Protein (YFP). CopB is a ∼50 kDa protein believed to have a critical role in chlamydial infection. 1 After adding a plasmid encoding YFP-CopB to an E. coli cell-free lysate, protein expression begins. We track the cell-free reaction over several hours using the EI-FLEX, a commercial instrument with FCS capability. As protein is expressed over time, YFP-CopB increases in concentration, and the EI-FLEX detects an increase in fluorescent signal above the background of the cell-free lysate. The FCS data collected gives information about the size, aggregation tendencies, rates of production and fluorescent protein maturation, and concentration of the YFP-CopB produced. The use of FCS concurrent with cell-free synthesis presents a simple method to characterize proteins of interest as they are produced without the need for purification.

Automated, modular assembly of reconstituted cell-free systems from in vitro-produced components.

Cell-free protein synthesis (CFPS) has emerged as a promising platform for point-of-care (POC) biosensing due to its programmability, rapid response, and ability to operate without living cells. Integrating CFPS into solid-state materials is essential for enabling long-term storage, portability, and spatial multiplexing required for practical POC applications. While previous efforts have demonstrated CFPS integration onto two-dimensional substrates such as paper, embedding CFPS machinery within a three-dimensional matrix that preserves its activity remains a significant challenge. Here, we propose a solid-state CFPS system comprised of a parylene-based scaffold loaded with CFPS machinery, i.e., CFPS in porous parylene (CinPP). The CinPP is fabricated through ice-templated chemical vapor deposition (CVD). We further demonstrate protein synthesis that is readily activated upon rehydration. The CinPP platform supports protein expression after prolonged storage and enables spatially localized and programmable expression of distinct fluorescent proteins. Furthermore, selective reconstitution of CFPS machinery with target-responsive genetic circuits demonstrates its potential as a modular sensing platform. This approach provides a versatile solid-state CFPS material that can be extended to programmable biosensors, portable diagnostic devices, and on-demand protein production systems.

Thermostable enzymes are highly desirable for industrial applications. Conventional thermostability screening typically relies on microplate assays; however, this approach becomes impractical as the library size increases exponentially with the number of mutated sites, resulting in excessive reagent use and high costs. Small droplet-based screening systems have recently emerged as an attractive alternative, offering reduced reagent consumption and scalability. However, constructing a heat-compatible screening system requires careful optimization of the reaction conditions under elevated temperatures. In this study, we applied our recently developed high-throughput screening system, which integrates hydrogel beads with cell-free protein synthesis (CFPS), to the selection of thermostable enzymes. Using two laccases with distinct thermostabilities, we showed that the hydrogel bead-based high-throughput screening system reliably discriminates the enzymes based on their thermal stability. The results highlight the potential of this platform as a cost-effective and scalable CFPS-based method for the directed evolution of thermostable enzymes.

Nucleic acid detection via protein readout through Cas-controlled gating of cell-free protein synthesis.

Construction of bio-layer interferometry biosensors via cell-free synthesized proteins for fishing bioactive compounds from Chinese herbs.

Adaptive laboratory evolution enhances methanol-driven CO2 fixation in Sporomusa sphaeroides KIAC.

Cell-free gene expression systems offer cell-like functionalities outside the confines of the cell, garnering increasing interest for applications from biomanufacturing to sensing. As applications expand, the need to implement economically scaled processes to produce cellular lysates grows. The protocols to produce these cellular lysates are complex, and the impact of altering many of the process variables remains understudied. Here, we set out to evaluate the effect of extended incubations at several points in the extract preparation process with the goal of identifying breakpoints that would enable flexibility in process implementation. As a model, we prepared lysates from 50 L cultures instead of typical 1 L volumes. We produced 72 lysates, 36 that were incubated overnight before and after culture centrifugation, and 36 that were incubated with and without a run-off reaction, each across different temperatures. We found that incubations before and after culture centrifugation substantially increased variability between culture replicates but did not reduce cell-free protein synthesis activity, contrary to conventional wisdom that materials should be kept cold as much as possible throughout the process. We also observed that omitting the run-off reaction reduced yields but resulted in lysates that were robust to incubation up to room temperature overnight. When a run-off reaction was included, activity dropped both as a function of duration and temperature, and the overall variability increased. Our work offers potential options for flexibility in implementing lysate production processes and motivates further investigation into how key processing steps relate to cell-free expression activity.

Limitations in global access to research and health care capacity undermine equity, sustainability, and resilience, particularly in resource-limited settings. Molecular diagnostics and biologic therapeutics are set to revolutionize medicine; however, our dependence on centralized biomanufacturing and cold chain logistics restricts access to these benefits. These constraints reflect a chronic and unmet global challenge. Here, with research teams in North and South America and Asia, we challenge the top-down paradigm by advancing community-driven solutions that empower underserved populations to participate in the bioeconomy, producing what they need, when, and where they need it. Our approach leverages low-burden biomanufacturing—built on cell-free protein synthesis and open-source hardware—to enable local, on-demand production of high-value bioproducts, including growth factors, vaccines, and diagnostic enzymes, demonstrating performance comparable to commercial gold standards. Implemented at 10 sites worldwide, this platform supported patient trials targeting globally relevant pathogens. Together, these efforts lay the foundation for a globally inclusive biomanufacturing ecosystem, where innovation goes beyond geographic boundaries.

Restriction endonuclease DpnI is a critical tool for eliminating parental templates in site-directed mutagenesis (SDM); however, the wild-type enzyme exhibits low efficiency at low substrate concentrations. In this study, we introduced a lysine mutation into the C-terminal winged helix (wH) domain of DpnI via structure-guided design to enhance electrostatic interactions and established a rapid screening platform using rolling circle amplification coupled with cell-free protein synthesis (CFPS). Results showed that the Q235K variant achieved a specific activity of 640 000 U/mg, 4-fold higher than the wild type, and increased the positive mutation rate in SDM from 82.7% to 92.3% while significantly reducing background plasmid residue. This study not only yielded a high-performance tool enzyme but also demonstrated the high efficiency of CFPS for rapid iteration and optimization in enzyme engineering.

Cell-free protein synthesis (CFPS) is a powerful and versatile platform that supports a wide range of applications, from fundamental studies of the genetic code to scalable and rapid protein production. The recently developed Pichia pastoris CFPS combines advantages of both prokaryotic and eukaryotic systems, including a rapid growth rate, inexpensive cultivation media, a well-established genetic toolbox, and the capability to perform post-translational modifications (PTMs). As such, it represents a promising alternative for both academic research and biopharmaceutical manufacturing. However, its broader application has been limited by relatively low protein yields and high reagent costs. In this study, building on a previously optimized reaction protocol, we further advanced the P. pastoris CFPS towards a more economical and efficient platform by reducing the cost of protein production. Through systematic screening of chemical additives and their combinations, we identified the most effective stabilizers and crowding agents to be incorporated in the reaction. Additionally, we applied a machine learning model to predict translation initiation rates and optimized the Kozak sequence for enhanced expression. We also evaluated lower-cost glycolytic intermediates as alternative substrates for ATP regeneration to reduce the cost of goods. Compared with the initial baseline condition using the unoptimized CrP/CrK system, the optimized system, incorporating a modified Kozak sequence and the addition of PEG-6000 and spermidine, resulted in a 10-fold increase in protein yield, while reducing the cost per gram of protein by 89%. This work underscores the importance of protein-stabilizing additives and the role of rationally designed DNA sequences with minimized mRNA structural complexity to enhance yield in CFPS. Our demonstration of glycolytic intermediates as a potential secondary energy system additionally provides the foundation for the development of a cost-effective P. pastoris CFPS.

Cell-free protein synthesis (CFPS) is an in vitro platform that enables rapid protein production using cell extracts, energy sources, and genetic templates. Owing to its fast response, elimination of cell culture, open reaction environment, lyophilization compatibility, and high programmability, CFPS has emerged as a versatile engine for diagnostic sensing. Recent advances have integrated CFPS with modular genetic circuits, CRISPR-based detection, isothermal amplification, and portable formats such as paper-based devices and microfluidic chips, enabling sensitive and specific detection of viral nucleic acids, pathogen antigens, and small-molecule targets. These platforms further support multiplexed and point-of-care testing, substantially reducing assay time, cost, and infrastructure requirements. Despite remaining challenges in biosensor design for novel targets, analytical sensitivity in complex samples, batch-to-batch reproducibility, and clinical translation, continued engineering optimization is rapidly improving CFPS performance and robustness. This review summarizes the fundamental principles of CFPS, its major technological platforms, recent progress in diagnostic applications, and key challenges and opportunities for future development.

Non-canonical amino acids (ncaas) are increasingly used in vaccinology to improve vaccine adaptability and immunogenicity. Cell-free protein synthesis (CFPS) offers a promising route for site-specific ncaa incorporation, but conventional prokaryotic CFPS systems show limitations to produce complex proteins requiring post-translational modifications while eukaryotic systems have historically been difficult to scale and show low protein yields. Here we establish efficient site-specific introduction of ncaas into complex proteins with the high-yielding and scalable eukaryotic tobacco BY-2 CFPS system (BYL), commercialized as ALiCE®. ncaa incorporation yields reached up to 2 mg/ml with linear scalability up to 10 ml. We applied ncaa incorporation in BYL to enable click chemistry bioconjugation of the receptor binding domain (RBD) of influenza hemagglutinin to pre-assembled hepatitis B core (HBc) virus-like particles (VLPs). The resulting VLP-RBD conjugates exhibited hemagglutination activity, unlike the individual components, and protected mice from weight loss after influenza challenge. This research thus enables ncaa introduction for recombinant proteins produced in BYL, constructing a novel plug-and-play vaccine platform and further expanding the capabilities of BYL to produce vaccine candidates and other proteins of interest.