Cell-free Protein Expression System Research Articles

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.

In-Hydrogel Cell-Free Protein Expression System as Biocompatible and Implantable Biomaterial.

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Engineering transmembrane signal transduction in synthetic membranes using two-component systems

Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step toward this goal, we investigated how bacterial two-component systems (TCSs) can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the TCS can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing.

Multiscale design of cell-free biologically active architectural structures.

Cell-free protein expression systems are here combined with 3D-printed structures to study the challenges and opportunities as biofabrication enters the spaces of architecture and design. Harnessing large-scale additive manufacturing of biological materials, we examined the addition of cell-free protein expression systems ("TXTL" i.e., biological transcription-translation machinery without the use of living cells) to printed structures. This allowed us to consider programmable, living-like, responsive systems for product design and indoor architectural applications. This emergent, pluripotent technology offers exciting potential in support of health, resource optimization, and reduction of energy use in the built environment, setting a new path to interactivity with mechanical, optical, and (bio) chemical properties throughout structures. We propose a roadmap towards creating healthier, functional and more durable systems by deploying a multiscale platform containing biologically-active components encapsulated within biopolymer lattices operating at three design scales: (i) supporting cell-free protein expression in a biopolymer matrix (microscale), (ii) varying material properties of porosity and strength within two-dimensional lattices to support biological and structural functions (mesoscale), and (iii) obtaining folded indoor surfaces that are structurally sound at the meter scale and biologically active (we label that regime macroscale). We embedded commercially available cell-free protein expression systems within silk fibroin and sodium alginate biopolymer matrices and used green fluorescent protein as the reporter to confirm their compatibility. We demonstrate mechanical attachment of freeze-dried bioactive pellets into printed foldable fibrous biopolymer lattices showing the first steps towards modular multiscale fabrication of large structures with biologically active zones. Our results discuss challenges to experimental setup affecting expression levels and show the potential of robust cell-free protein-expressing biosites within custom-printed structures at scales relevant to everyday consumer products and human habitats.

The role of lipid bilayer composition in cell-free expressed ABC transporter folding.

Cell-free protein expression systems provide a powerful tool to mechanistically probe how membrane composition, and the resulting biophysical properties, affect membrane protein (MP) complex folding and function. These systems can be designed without translocons, insertases, and chaperones commonly used to assist in membrane protein folding to isolate the impact of membrane properties on the folding process. In this study we investigate the ABC transporter, SapABCDF, in the gram-negative non-typeable Haemophilus influenzae (NTHI). This transporter sequesters heme and antimicrobial peptides (AMPs) from the periplasm into the cytoplasm via ATPase activity and understanding factors that contribute to MP complex folding and function is critical for developing antimicrobial treatments for NTHI and reduce the severity of infection. Using a cell-free protein expression system, we expressed each protein in the SapABCDF transporter in the presence of small unilamellar lipid vesicles (SUVs). We developed a sucrose flotation assay to evaluate protein colocalization to fluorescently labeled SUVs after cell-free expression and found that all components of the SapBCDF system express and colocalize to vesicles, suggesting their membrane integration and assembly. We then conducted a co-translational MP folding assay with a C-terminal mEGFP tag to determine membrane compositions that promote MP folding and expression. Analysis of cell-free expression products via western blot suggests that one transmembrane domain, SapC, expresses preferentially into SUVs containing phosphatidylethanolamine (PE). Ongoing studies aim to characterize transporter functionality and biophysically characterize the impact of bilayer biophysical properties on MP folding. Together, our results should improve our understanding of the biophysical requirements for properly folded and functioning ABC transporters.

Tuning the composition of supported lipid bilayers in a QCMD to uncover rules of membrane protein incorporation.

Cells are not very cooperative when it comes to helping us understand them better. They are small, needy, complex, and they have their own lives to run. To circumvent these flaws of cells our lab has developed the all E. coli cell-free protein expression system called TXTL. TXTL can express genes and execute genetic programs in-vitro using the transcriptional and translational machinery of E. coli cells without the E. coli cells themselves. So far with TXTL our lab has probed both fundamental and applied ideas in gene regulation, metabolism, self-assembly, and encapsulation.

Characterization of Integrase and Excisionase Activity in a Cell-Free Protein Expression System Using a Modeling and Analysis Pipeline.

We present a full-stack modeling, analysis, and parameter identification pipeline to guide the modeling and design of biological systems starting from specifications to circuit implementations and parametrizations. We demonstrate this pipeline by characterizing the integrase and excisionase activity in a cell-free protein expression system. We build on existing Python tools─BioCRNpyler, AutoReduce, and Bioscrape─to create this pipeline. For enzyme-mediated DNA recombination in a cell-free system, we create detailed chemical reaction network models from simple high-level descriptions of the biological circuits and their context using BioCRNpyler. We use Bioscrape to show that the output of the detailed model is sensitive to many parameters. However, parameter identification is infeasible for this high-dimensional model; hence, we use AutoReduce to automatically obtain reduced models that have fewer parameters. This results in a hierarchy of reduced models under different assumptions to finally arrive at a minimal ODE model for each circuit. Then, we run sensitivity analysis-guided Bayesian inference using Bioscrape for each circuit to identify the model parameters. This process allows us to quantify integrase and excisionase activity in cell extracts enabling complex-circuit designs that depend on accurate control over protein expression levels through DNA recombination. The automated pipeline presented in this paper opens up a new approach to complex circuit design, modeling, reduction, and parametrization.

Rapid and Finely-Tuned Expression for Deployable Sensing Applications.

Organisms from across the tree of life have evolved highly efficient mechanisms for sensing molecules of interest using biomolecular machinery that can in turn be quite valuable for the development of biosensors. However, purification of such machinery for use in in vitro biosensors is costly, while the use of whole cells as in vivo biosensors often leads to long sensor response times and unacceptable sensitivity to the chemical makeup of the sample. Cell-free expression systems overcome these weaknesses by removing the requirements associated with maintaining living sensor cells, allowing for increased function in toxic environments and rapid sensor readout at a production cost that is often more reasonable than purification. Here, we focus on the challenge of implementing cell-free protein expression systems that meet the stringent criteria required for them to serve as the basis for field-deployable biosensors. Fine-tuning expression to meet these requirements can be achieved through careful selection of the sensing and output elements, as well as through optimization of reaction conditions via tuning of DNA/RNA concentrations, lysate preparation methods, and buffer conditions. Through careful sensor engineering, cell-free systems can continue to be successfully used for the production of tightly regulated, rapidly expressing genetic circuits for biosensors.

Rapid engineering of surface acoustic wave devices using cell-free expressed bitter receptors towards chemical sensing

The engineering of devices with various functional molecules in a high-efficient and convenient manner is crucial for biosensors towards biomedical applications. In this study, a multi-channel surface acoustic wave (SAW) device was rapid engineered with bitter receptors prepared from a cell-free protein expression system for the development of biomimetic biosensors for the detection of bitter substances. The novelty of this study mainly attributed to the utilization of DNA-directed immobilization (DDI) method to achieve high-efficient and controllable immobilization of bitter receptors onto sensitive area of SAW device. SAW device is employed as a transducer to monitor the specific interactions between bitter receptors and their ligands, which is a mass-sensitive device that can monitor tiny mass changes by recording the changes in the phase shift of SAW. The obtained results indicated that this biomimetic biosensor was able to respond to different bitter substances by generating various responsive signals, which show good stability. In addition, the distinct responsive patterns could be used to discriminating different bitter substances. It was successfully used to detect and discriminate 10 μM MgSO4, 0.1 μM denatonium, 0.1 μM D-(−)-Salicin, and 0.1 μM quinine. This biomimetic biosensor has great potential to be used as a novel tool for chemical sensing as well as for the research of mechanisms of bitter receptor interacting with their ligands.

A Functional Carbohydrate Degrading Enzyme Potentially Acquired by Horizontal Gene Transfer in the Genome of the Soil Invertebrate Folsomia candida.

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage.

The Escherichia coli DnaK chaperone stimulates the α-complementation of β-galactosidase.

pUC18 and pUC19 are well-known high copy-number plasmid vectors routinely used for DNA cloning purposes. We show here that, in Escherichia coli transformed by native pUC18, the α-complementation of β-galactosidase (i.e., mediated by the peptide LacZα18) is intrinsically weak and slow, but is greatly stimulated by the DnaK/DnaJ/GrpE chaperone system. In contrast, the α-complementation mediated by the peptide LacZα19 (in E. coli transformed by the native pUC19) is much more efficient and therefore does not require the assistance of the DnaK chaperone machinery. The marked difference between these two LacZα peptides is reproduced in a cell-free protein expression system coupled with α-complementation. We conclude that: (i) α-complementation of β-galactosidase is DnaK-mediated depending upon the LacZα peptide donor; (ii) DnaK, sensu stricto, is not necessary for α-complementation, but can enhance it to a great extent; (iii) this observation could be used to establish an easy and inexpensive method for screening small molecules libraries in search of DnaK inhibitors and also for deciphering the DnaK-mediated protein quality control mechanism.

Rapid and Facile Preparation of Giant Vesicles by the Droplet Transfer Method for Artificial Cell Construction.

Giant vesicles have been widely used for the bottom-up construction of artificial (or synthetic) cells and the physicochemical analysis of lipid membranes. Although methods for the formation of giant vesicles and the encapsulation of molecules within them have been established, a standardized protocol has not been shared among researchers including non-experts. Here we proposed a rapid and facile protocol that allows the formation of giant vesicles within 30 min. The quality of the giant vesicles encapsulating a cell-free protein expression system was comparable to that of the ones formed using a conventional method, in terms of the synthesis of both soluble and membrane proteins. We also performed protein synthesis in artificial cells using a lyophilized cell-free mixture and showed an equivalent level of protein synthesis. Our method could become a standard method for giant vesicle formation suited for artificial cell research.

Characterization of phosphorylation status and kinase activity of Src family kinases expressed in cell-based and cell-free protein expression systems

Characterization of phosphorylation status and kinase activity of Src family kinases expressed in cell-based and cell-free protein expression systems

Applications of Cell-Free Synthesized Membrane Protein Precipitates.

Cell-free protein expression systems are new core platforms for membrane protein synthesis. Expression in thepresence of supplied artificial hydrophobic environments such as nanomembranes or micelles allows the co-translational solubilization and folding of membrane proteins. In theabsence 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 avery 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 techniquesin 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.

Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems.

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery.

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red‐flowered gentian

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected invitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

DiRect: Site-directed mutagenesis method for protein engineering by rational design

Site-directed mutagenesis (SDM), an indispensable method in molecular biology and protein engineering, is rather time-consuming and laborious. Protein engineering, especially that of enzymes, nowadays increasingly relies on rational design approaches in which both SDM and protein expression are the bottlenecks because they are generally based on the recombinant DNA technology. Here, we developed a new PCR-based mutagenesis method, DiRect, that achieves high performance in product quality (≥99% substitution) without recombinant DNA technology. We applied DiRect in combination with a cell-free protein expression system to an industrially relevant enzyme, nicotinamide adenine dinucleotide phosphate-dependent 3-quinuclidinone reductase from Rhodotorula rubra. In a single round of screening, 90 newly designed mutant proteins were produced within two days, and an unreported mutant (Q135I) exhibiting much higher thermostability than the wild-type enzyme was successfully identified within one extra day. Thus, DiRect is a simple, efficient, and potentially scalable SDM method.

Construction of an Artificial Cell Capable of Protein Expression at Low Temperatures Using a Cell Extract Derived from Pseudomonas fluorescens

A liposome-based artificial cell (LBAC) consists of a liposome encapsulating a cell-free protein expression system (CFPES) and protein-encoding DNA. It is surrounded by a lipid bilayer membrane and synthesizes proteins that resemble actual cells. Hence, they have been one of the most studied artificial cells. According to recent studies, they have been able to sense bio-functional molecules by synthesizing fluorescent proteins in response to target molecules. Therefore, they are expected to be used as biosensors. However, previously reported LBACs encapsulated the CFPES derived from Escherichia coli, resulting in the most productive protein expression at 20–40 °C. To broaden the range of their working temperatures to lower temperatures, in this study, we constructed LBACs using a CFPES derived from Pseudomonas fluorescens that grows at a temperature range of 4 °C to 30 °C. We then demonstrated that the constructed LBAC expressed proteins at 8 °C and that, the protein expression capability of the LBAC derived from P. fluorescens was four-fold higher than that derived from E. coli at 8 °C. This study will pave the way for the development of artificial cell-based biosensors that work in cold environments or for the synthesis of heat-labile proteins in LBACs.