Cell-free Protein Expression Research Articles

Cofactor recycling strategies for secondary metabolite production in cell-free protein expression systems

Cofactor recycling strategies for secondary metabolite production in cell-free protein expression systems

Genetically programmed synthetic cells for thermo-responsive protein synthesis and cargo release

Synthetic cells containing genetic programs and protein expression machinery are increasingly recognized as powerful counterparts to engineered living cells in the context of biotechnology, therapeutics and cellular modelling. So far, genetic regulation of synthetic cell activity has been largely confined to chemical stimuli; to unlock their potential in applied settings, engineering stimuli-responsive synthetic cells under genetic regulation is imperative. Here we report the development of temperature-sensitive synthetic cells that control protein production by exploiting heat-responsive mRNA elements. This is achieved by combining RNA thermometer technology, cell-free protein expression and vesicle-based synthetic cell design to create cell-sized capsules able to initiate synthesis of both soluble proteins and membrane proteins at defined temperatures. We show that the latter allows for temperature-controlled cargo release phenomena with potential implications for biomedicine. Platforms like the one presented here can pave the way for customizable, genetically programmed synthetic cells under thermal control to be used in biotechnology.

PP01.23 Plasma cell-free RNA PD-L1 or Tissue PD-L1 Protein Expression and Immunotherapy Outcomes in Advanced Non-Small Cell Lung Cancer

PP01.23 Plasma cell-free RNA PD-L1 or Tissue PD-L1 Protein Expression and Immunotherapy Outcomes in Advanced Non-Small Cell Lung Cancer

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.

Circular single-stranded DNA as a programmable vector for gene regulation in cell-free protein expression systems

Cell-free protein expression (CFE) systems have emerged as a critical platform for synthetic biology research. The vectors for protein expression in CFE systems mainly rely on double-stranded DNA and single-stranded RNA for transcription and translation processing. Here, we introduce a programmable vector - circular single-stranded DNA (CssDNA), which is shown to be processed by DNA and RNA polymerases for gene expression in a yeast-based CFE system. CssDNA is already widely employed in DNA nanotechnology due to its addressability and programmability. To apply above methods in the context of synthetic biology, CssDNA can not only be engineered for gene regulation via the different pathways of sense CssDNA and antisense CssDNA, but also be constructed into several gene regulatory logic gates in CFE systems. Our findings advance the understanding of how CssDNA can be utilized in gene expression and gene regulation, and thus enrich the synthetic biology toolbox.

Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action.

Chemicals bacteria encounter at the infection site could shape their stress and antibiotic responses; such effects are typically undetected under standard lab conditions. Polyamines are small molecules typically overproduced by the host during infection and have been shown to alter bacterial stress responses. We sought to determine the effect of polyamines on the antibiotic response of Klebsiella pneumoniae, a Gram-negative priority pathogen. Interestingly, putrescine and other natural polyamines sensitized K. pneumoniae to azithromycin, a macrolide protein translation inhibitor typically used for Gram-positive bacteria. This synergy was further potentiated in the physiological buffer, bicarbonate. Chemical genomic screens suggested a dual mechanism, whereby putrescine acts at the membrane and ribosome levels. Putrescine permeabilized the outer membrane of K. pneumoniae (NPN and β-lactamase assays) and the inner membrane (Escherichia coli β-galactosidase assays). Chemically and genetically perturbing membranes led to a loss of putrescine-azithromycin synergy. Putrescine also inhibited protein synthesis in an E. coli-derived cell-free protein expression assay simultaneously monitoring transcription and translation. Profiling the putrescine-azithromycin synergy against a combinatorial array of antibiotics targeting various ribosomal sites suggested that putrescine acts as tetracyclines targeting the 30S ribosomal acceptor site. Next, exploiting the natural polyamine-azithromycin synergy, we screened a polyamine analogue library for azithromycin adjuvants, discovering four azithromycin synergists with activity starting from the low micromolar range and mechanisms similar to putrescine. This work sheds light on the bacterial antibiotic responses under conditions more reflective of those at the infection site and provides a new strategy to extend the macrolide spectrum to drug-resistant K. pneumoniae.

Production of a covalent conjugate vaccine using cell-free protein expression (XpressCF™) and click chemistry

Abstract Chlamydia trachomatis is the most common bacterial sexually transmitted infection globally and a vaccine is urgently needed. To address this, we determined that Chlamydia protease-like activity factor (CPAF) is the most immunoprevalent and immunodominant antigen for CD4 T cells and B cells in Chlamydia exposed women and mice. CPAF is a 70-kDa serine protease that is secreted into the host cell cytoplasm and subsequently released into the extracellular milieu where it paralyzes neutrophils to evade phagocytosis. CPAF contains a fold similar to other known serine proteases and is processed into C- and N-terminal fragments that heterodimerize. To improve and facilitate vaccine scale-up and immunogenicity, we developed CPAF variants incorporating non-native amino acids at specific sites using the proprietary cell free expression platform XpressCF™. These CPAF variants were covalently conjugated to DBCO-derivatized adjuvants using click chemistry to make novel antigen-adjuvant conjugate vaccines. Site-specific conjugation enables important T- and B-cell epitopes to be avoided for better immune recognition and provides a method to incorporate a specific number of conjugated adjuvants. These conjugated vaccines provide a means to simultaneously deliver antigen and adjuvant and potentially reduce the amount of adjuvant required for vaccine efficacy leading to fewer side effects and dose sparing.

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.

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.

In vitro co-expression chromatin assembly and remodeling platform for plant histone variants

Histone variants play a central role in shaping the chromatin landscape in plants, yet, how their distinct combinations affect nucleosome properties and dynamics is still largely elusive. To address this, we developed a novel chromatin assembly platform for Arabidopsis thaliana, using wheat germ cell-free protein expression. Four canonical histones and five reported histone variants were used to assemble twelve A. thaliana nucleosome combinations. Seven combinations were successfully reconstituted and confirmed by supercoiling and micrococcal nuclease (MNase) assays. The effect of the remodeling function of the CHR11-DDR4 complex on these seven combinations was evaluated based on the nucleosome repeat length and nucleosome spacing index obtained from the MNase ladders. Overall, the current study provides a novel method to elucidate the formation and function of a diverse range of nucleosomes in plants.

Plasma cell-free RNA PD-L1 or tissue PD-L1 protein expression and outcomes with first-line immunotherapy in metastatic non-small cell lung cancer

BackgroundTissue programmed death ligand-1 (PD-L1) protein expression is associated with immune checkpoint inhibitor (ICI) treatment benefit in metastatic non-small cell lung cancer (NSCLC). However, tissue PD-L1 protein testing is limited by sampling tumor heterogeneity and fraught with tissue acquisition difficulties. A liquid biopsy-based PD-L1 assay could overcome these limitations of tissue PD-L1 testing. MethodsAn observational cohort of patients with metastatic NSCLC treated with first-line ICI-based therapies were retrospectively assessed for a pre-planned endpoint of median and 3-year landmark overall survival (OS) based upon plasma cell-free RNA (cfRNA) PD-L1 expression by a commercial exosome-free real-time qPCR assay or tissue PD-L1 protein expression (Dako 22C3) performed in CLIA/CAP accredited laboratories. Results53 contemporaneous patients in 3 patient cohorts were compared with a median follow-up 34 months. 16 patients were cfRNA plasma PD-L1 positive, including 6 (37 %) tissue PD-L1 negative or tissue quantity not sufficient for testing; 16 were plasma PD-L1 negative but tissue PD-L1 positive; 21 were both plasma and tissue PD-L1 negative. OS was identical whether positive plasma cfRNA PD-L1 expression or positive tissue PD-L1 protein expression (median OS 15 months; 3-year landmark OS 30 %; hazard ratio (HR) 0.97; 95 % CI, 0.44–2.10; p-value = 0.93). Within the positive plasma PD-L1 cohort there was no differing OS whether tissue PD-L1 positive, negative, or unknown (median OS 15 months; 3-year landmark OS 30 %; HR 1.15; 95 % CI, 0.322–4.05; p-value = 0.81). Positive plasma cfRNA expression was associated with a numerically longer median and higher 3-year OS compared to patients lacking PD-L1 expression (median 15 months versus 8 months; 3-year landmark OS 30 % versus 15 %; HR 0.57, 95 % CI 0.26–1.20; p-value = 0.11). ConclusionsIn this retrospective study of real-world metastatic NSCLC patients, plasma cfRNA PD-L1 expression was similarly predictive of ICI benefit as tissue PD-L1 protein expression.

DnaK promotes autophosphorylation of DYRK1A and its family kinases in Escherichia coli-based cell-free protein expression

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is one of the drug target kinases involved in neurological disorders. DYRK1A phosphorylates substrate proteins related to disease progression in an intermolecular manner. Meanwhile, DYRK1A intramolecularly phosphorylates its own residues on key segments during folding process, which is required for its activation and stabilization. To reproduce the autophosphorylation in vitro, DYRK1A was expressed in Escherichia coli-based cell-free protein synthesis system. Although this system was useful for investigating autophosphorylation of serine residue at position 97 (Ser97) in DYRK1A, only a small fraction of the synthesized protein was successfully autophosphorylated. In this study, we found that the addition of DnaK, a bacterial HSP70 chaperone, to cell-free expression of DYRK1A promoted its Ser97 autophosphorylation. Structure prediction with AlphaFold2 indicates that Ser97 forms a hydrogen bond within an α-helix structure, indicating a possibility that DnaK unfolds the α-helix and maintains the structure around Ser97 in a conformation susceptible to phosphorylation. In addition, DnaK promoted phosphorylation of DYRK1B and HIPK2, but not DYRK2 and DYRK4, suggesting a sequence selectivity in the action of DnaK. This study provides a facile method for promoting autophosphorylation of DYRK family kinases in cell-free protein expression.

Plasmid Crosstalk in Cell-Free Expression Systems.

Although cell-free protein expression has been widely used for the synthesis of single proteins, cell-free synthetic biology has rapidly expanded to new, more complex applications. One such application is the prototyping or implementation of complex genetic networks involving the expression of multiple proteins at precise ratios, often from different plasmids. However, expression of multiple proteins from multiple plasmids may inadvertently result in unexpected, off-target changes to the levels of the proteins being expressed, a phenomenon termed plasmid crosstalk. Here, we show that the effects of plasmid crosstalk─even at the qualitative level of increases vs decreases in protein expression─depend on the concentration of plasmids in the reaction and the type of transcriptional machinery involved in the expression. This crosstalk can have a significant impact on genetic circuitry function and even interpretation of simple experimental results and thus should be taken into consideration during the development of cell-free applications.

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.

Lipid dependence of the conformational coupling across the membrane bilayer of full-length epidermal growth factor receptor.

Membrane proteins regulate several vital cellular processes, establishing them as major drug targets. A main function of membrane proteins is to transduce extracellular signal across the membrane bilayer. One such protein is the epidermal growth factor receptor (EGFR), which plays a crucial role in cell signaling and is implicated in cancer. While the structures of the extracellular and intracellular regions of this protein have been well-elucidated individually, conformational coupling connecting these two regions during signal transduction is challenging to probe due to the mixture of hydrophobic and hydrophilic domains intrinsic to membrane proteins. Here, I will discuss the signal transduction mechanism across full-length EGFR. We combine two biochemical tools, cell-free protein expression and nanodiscs, to isolate full-length, functional EGFR in a near-physiological environment. Using a multidisciplinary approach involving single-molecule Förster resonance energy transfer, mutagenesis, and molecular dynamics simulations, we observe a compaction in the intracellular domain of EGFR upon extracellular ligand binding in a neutral lipid environment. The ligand-induced extracellular/intracellular conformational coupling is reversed in the presence of anionic lipids, robust around the physiological 15-30% anionic content, and disappears in the presence of cholesterol. Our findings show how the extracellular and intracellular domains are coupled to each other in this critical receptor and the impact of the lipid composition in this conformational coupling. Our results could be universal to other membrane receptors which share the same structural homology and perform other significant functions.

Unraveling the key-lock interaction mechanisms of the human neuropeptide Y type 4 receptor (hY4R) via solid-state NMR spectroscopy.

Human neuropeptide Y receptors (hYxR) are rhodopsin-like, peptide-binding G-protein coupled receptors (GPCRs). They are expressed within the gut-brain axis network, which allows several possible activation mechanisms, and thus downstream signaling pathways. hYxR therefore regulate a broad spectrum of biological functions (e.g., metabolic balance, vasoconstriction/dilation, circadian rhythm), and also play a central role in the development of several wide-spread pathologies such as several types of cancer, obesity/anorexia, anxiety and depression. Their functions and dysfunctions are tightly dependent on the specific extra- and intracellular coupling partners, as well as on the type of lipid membrane in which they are embedded. Understanding this complex interplay of receptor conformations/bound ligands/lipid environment has received much attention in the biochemical and biophysical communities, especially over the last 20 years, often motivated by the urgent need to develop selective therapeutics, based on receptor-specific lock-key mechanisms. Here, we present our novel approach to the study of conformational dynamics of the human Y receptor subtype 4 (hY4R - the lock), which has received very little attention compared to the rest of the hYxR family. Using cell-free protein expression strategies and solid-state NMR spectroscopy, we characterize structural changes of the receptor at the molecular level, when activated by different endogenous peptide ligands (the keys), namely pancreatic polypeptide (PP) and neuropeptide Y (NPY). This approach has the enormous potential to bringing us one step closer to understanding the specific key-lock interactions, which give rise to particular biological functions, and thus to the development of subtype-specific therapeutics.

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.