Cell-free Synthesis Research Articles

Functional discovery and production technology for natural bioactive peptides

Bioactive peptides play important roles in promoting human health, such as lowering blood pressure, blood sugar and blood lipid, anti-obesity, and anti-cancer. Thus, exploring functional bioactive peptides and developing efficient production technologies are of crucial importance. Herein, we review the development of function discovery and production technology for natural bioactive peptides. Presently, the top-down and bottom-up approaches are mainly used for the function discovery and production of natural active peptides. The top-down approach includes the direct extraction and identification for functional discovery, and the direct extraction, enzymatic hydrolysis and microbial fermentation for production. The bottom-up approach includes the polypeptide modification and database mining for functional discovery, and the chemical synthesis, enzyme synthesis, recombinant expression and cell-free synthesis for production. The top-down approach is usually associated with complicated process, lower efficiency, higher cost, harder quality control, and uncertain functionality, while the bottom-up approach is more suitable for the development of peptide drugs but difficult to be used for functional foods. With the technology development of sequencing and mass spectrometry, it is easier to obtain the proteomic information of various organisms at the molecular level. Based on the proteomic information, the top-down and bottom-up approaches can be combined to overcome the disadvantages of using these two approaches alone, thus providing a new strategy for the rapid development and production of natural active peptides.

A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)–specific monoclonal antibody (mAb), the Escherichia coli–expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli–expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient–derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes.Key points• The antibody showed cross-reactivity with capsid proteins of different hepeviruses.• The linear epitope of the antibody was mapped in a partially surface-exposed region.• The antibody detected native HEV-3 antigen in infected mammalian cells.

Engineering molecular translation systems.

Molecular translation systems provide a genetically encoded framework for protein synthesis, which is essential for all life. Engineering these systems to incorporate non-canonical amino acids (ncAAs) into peptides and proteins has opened many exciting opportunities in chemical and synthetic biology. Here, we review recent advances that are transforming our ability to engineer molecular translation systems. In cell-based systems, new processes to synthesize recoded genomes, tether ribosomal subunits, and engineer orthogonality with high-throughput workflows have emerged. In cell-free systems, adoption of flexizyme technology and cell-free ribosome synthesis and evolution platforms are expanding the limits of chemistry at the ribosome's RNA-based active site. Looking forward, innovations will deepen understanding of molecular translation and provide a path to polymers with previously unimaginable structures and functions.

Biosynthetic Strategies for Macrocyclic Peptides.

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.

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

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

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

THE USE OF A CELL-FREE PROTEIN SYNTHESIS FOR OBTAINING BACTERIAL DIGUANYLATCYCLASE AND TWO CHIMERIC PROTEINS

The novel promising trend of biotechnology is cell-free synthesis of proteins. Possibility of producing two chimeric proteins: adenosine deaminase of Escherichia coli fused with human annexin-A5, DNA-affine domain of bacteria Sulfolobus solfataricus fused with modified Taq-DNA-polymerase and bacterial enzyme diguanylatecyclase of Thermotoga maritima, by cell-free synthesis procedure as an alternative to conventional cultivation of microbial strains-producers in a bioreactor was studied in this research. Chimeric RNA polymerase of T7 bacteriophage, S30-cell extract of E. coli and multicopy plasmid pET42mut were engaged for protein synthesis by cell-free protein synthesis system. The first successful production of these proteins was demonstrated
 in CFPS system.

Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity.

Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P < 0.0001]), faster patient recovery (at day 14, 93.9% in the enisamium group had recovered versus 32.5% in placebo group [P < 0.0001]), and reduced disease symptoms (from 9.6 ± 0.7 to 4.6 ± 0.9 score points in enisamium group versus 9.7 ± 1.1 to 5.6 ± 1.1 score points in placebo group [P < 0.0001]) compared to those in the placebo group. Using mass spectrometry, and cell-based and cell-free viral RNA synthesis assays, we identified a hydroxylated metabolite of enisamium, VR17-04. VR17-04 is capable of inhibiting influenza virus RNA synthesis and is present in plasma of patients treated with enisamium. VR17-04 inhibits the activity of the influenza virus RNA polymerase more potently than its parent compound. Overall, these results suggest that enisamium is metabolized in humans to an inhibitor of the influenza virus RNA polymerase that reduces viral shedding and improves patient recovery in influenza patients. (This study has been registered at ClinicalTrials.gov under identifier NCT04682444.).

Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer.

Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.

Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging.

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. GdIII -GdIII distances measured by double electron-electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-GdIII were indistinguishable from GdIII -GdIII distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.

Process economics evaluation of cell-free synthesis for the commercial manufacture of antibody drug conjugates.

Continuous improvements of cell-free synthesis (CFS) systems have generated interest in adopting the technology for the manufacture of biologics. This paper provides an evaluation of the manufacturing cost-effectiveness of CFS for the commercial production of antibody-drug conjugates (ADCs). The evaluation was performed using an advanced techno-economic engine (TEE) built in Python. The TEE is programmed in an object-oriented environment capable of simulating a plethora of process flowsheets and predicting size and cost metrics for the process and the facility. A case study was formulated to compare the economics of whole bioprocesses based on either a CFS system or a mammalian cell system (CHO) for the manufacture of an ADC at a range of product demands. The analysis demonstrated the potential of CFS for the commercial manufacture of biologics and identified key cost drivers related to the system. The CFS system showed an approximately 80% increase in the cost of goods compared to CHO with a significant cost attributed to the in-house manufacture of the bacterial cell extract, necessary for the CFS reaction step in the process. A sensitivity and target analysis highlighted the need for further process improvements especially in the titer for the CFS process to become more competitive against well-established systems.

In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity

The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3 × 10–3 and 13.2 × 10–3, depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in E. coli. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system.

Synthesis of Fluorescently Labeled Antibodies Using Non-Canonical Amino Acids in Eukaryotic Cell-Free Systems.

Cell-free protein synthesis (CFPS) enables the development of antibody conjugates, such as fluorophore conjugates and antibody-drug conjugates (ADCs), in a rapid and straightforward manner. In the first part, we describe the cell-free synthesis of antibodies containing fluorescent non-canonical amino acids (ncaa) by using pre-charged tRNA. In the second part, we describe the cell-free synthesis of antibodies containing ncaa by using an orthogonal system, followed by the site-specific conjugation of the fluorescent dye DyLight 650-phosphine. The expression of the antibodies containing ncaa was analyzed by SDS-PAGE, followed by autoradiography and the labeling by in-gel fluorescence. Two different fluorescently labeled antibodies could be generated.

Strategy exploration for developing robust lyophilized cell-free systems

Cell-free protein synthesis system is emerging as a powerful tool for rapid expression of therapeutic proteins. However, one drawback of cell-free technology is the necessity to store the major components below freezing in bulky aqueous solutions. To preserve the cell-free synthesis system for a longer time, the lyophilized cell-free system was developed, and its stability under freeze-drying conditions was explored. The novelty and the difference of this study from the others lie in determining more detailed storage conditions, which could provide a more accurate storage strategy. The effects of the treatment time, the storage temperature, and the component-mixing mode on the freeze-dried cell-free system were investigated. This study explored a robust storage strategy for freeze-dried cell-free systems and could further open up a better opportunity for on-demand synthesis of therapeutic proteins.

Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins.

Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins) with multiple intramolecular disulphide bonds which are typically difficult to express in a soluble and folded state in the reducing environments found inside a cell. In some cases, the inclusion of a recombinant disulphide isomerase DsbC further enhances the expression levels of correctly folded hydrophobins. Using this protocol, we can achieve milligram levels of protein expression per ml of reaction. While our target proteins are the fungal hydrophobins, it is likely that this protocol with some minor variations can be used to express other proteins with multiple intramolecular disulphide bonds in a natively folded state. Graphic abstract: Figure 1.Workflow for cell-free protein expression and single-step purification using affinity chromatography. A. E. coli S30 lysate prepared as described in Apponyi et al. (2008) can be stored for up to several years at -80°C without any loss of activity in our experience. B. The S30 lysate, plasmid DNA that encodes for the protein of interest along with an affinity tag and components required for transcription and translation are added to the reaction mix. Following a single-step protein purification, the protein of interest can be isolated for further use.

Converging conversion – using promiscuous biocatalysts for the cell-free synthesis of chemicals from heterogeneous biomass

Mixed sugars are often found in biomass hydrolysis. Promiscuous biocatalysts are crucial for stereo-convergent synthesis of chemicals from biomass. A “plug-and-play” strategy allowed us to divert the production of a chemical of interest from two pentoses.

A Sustainable and Efficient Artificial Microgel System: Toward Creating a Configurable Synthetic Cell

Artificial cells are a powerful platform in the study of synthetic biology and other valuable fields. They share a great potential in defining and utilizing the superiority of the living system. Here, a protein synthesis system based on thermal responsive hydrogels with porous structure is reported. The hydrogels can immobilize plasmids on the surface inside their porous structure through a volume phase transition upon 34 °C, forming an aggregation state of DNAs as in nature conditions. The artificial microgels can carry out bioreactions in cell-free systems and exhibit a sustainable and efficient performance for protein translation. The protein synthesis level reaches a maximum of twice more than that in a conventional solution system when the plasmid concentration is 10-20ng µL-1 , along with a doubled effective interval. This is perhaps attributed to confined transcription and translation processes in the near-surface area of hydrogels. Summarily, the research provides an easy-handling approach in fabricating effective microgels for cell-free synthesis and also inspirations for constructing a configurable artificial cell.

Cell-free systems for accelerating glycoprotein expression and biomanufacturing.

Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic N-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines.

In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies.

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membraneproteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468kDa TET2 protein assembly for NMR investigations is presented.

Cell-Free Synthesis of Natural Compounds from Genomic DNA of Biosynthetic Gene Clusters.

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.