Cell-free Synthesis Research Articles

Cell-Free Synthesis Goes Electric: Dual Optical and Electronic Biosensor via Direct Channel Integration into a Supported Membrane Electrode.

Assembling transmembrane proteins on organic electronic materials is one promising approach to couple biological functions to electrical readouts. A biosensing device produced in such a way would enable both the monitoring and regulation of physiological processes and the development of new analytical tools to identify drug targets and new protein functionalities. While transmembrane proteins can be interfaced with bioelectronics through supported lipid bilayers (SLBs), incorporating functional and oriented transmembrane proteins into these structures remains challenging. Here, we demonstrate that cell-free expression systems allow for the one-step integration of an ion channel into SLBs assembled on an organic conducting polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). Using the large conductance mechanosensitive channel (MscL) as a model ion channel, we demonstrate that MscL adopts the correct orientation, remains mobile in the SLB, and is active on the polyelectrolyte surface using optical and electrical readouts. This work serves as an important illustration of a rapidly assembled bioelectronic platform with a diverse array of downstream applications, including electrochemical sensing, physiological regulation, and screening of transmembrane protein modulators.

Electrochemical studies of the mitochondrial ROMK2 potassium channel activity reconstituted into the free-standing and tethered bilayer lipid membranes

The renal-outer-medullary‑potassium (ROMK2) channel modulates potassium transport in the kidney. It has been postulated that the ROMK2 is the pore-forming subunit of the mitochondrial ATP-sensitive potassium channel as a mediator of cardioprotection. In this study, cell-free synthesis of the ROMK2 was performed in presence of membrane scaffold protein (MSP1D1) nanodiscs. Activity measurements were achieved after channel reconstitution into the planar lipid bilayer and tethered bilayer lipid membranes. Both methods allowed for monitoring of channel function, verified with channel blocking and activation/re-activation experiments. The primary function of the mitochondrial potassium channels is to regulate the potential of the mitochondrial membrane, which allows them to play an important role in cytoprotection. This work focuses on obtaining the ROMK2 using a cell-free expression system, followed by the incorporation of the channel protein into the lipid bilayer and studying the influence of voltage changes and molecular modulators on channel activity. Channel activity was measured after its reconstitution into two models of lipid bilayers - BLM (Bilayer Lipid Membrane) and tBLM (Tethered Bilayer Lipid Membrane) deposited on a solid gold electrode. These two model membranes and electrochemical measurements made it possible to measure the flux of K+ ions in the presence of channel modulators.

Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes.

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) KV11.1 and the voltage-gated sodium channel hNaV1.5.

Evaluation of Cell-Free Synthesized Human Channel Proteins for In Vitro Channel Research.

Despite channel proteins being important drug targets, studies on channel proteins remain limited, as the proteins are difficult to express and require correct complex formation within membranes. Although several in vitro synthesized recombinant channels have been reported, considering the vast diversity of the structures and functions of channel proteins, it remains unclear which classes of channels cell-free synthesis can be applied to. In this study, we synthesized 250 clones of human channels, including ion channel pore-forming subunits, gap junction proteins, porins, and regulatory subunits, using a wheat cell-free membrane protein production system, and evaluated their synthetic efficiency and function. Western blotting confirmed that 95% of the channels were successfully synthesized, including very large channels with molecular weights of over 200 kDa. A subset of 47 voltage-gated potassium ion channels was further analyzed using a planar lipid bilayer assay, out of which 80% displayed a voltage-dependent opening in the assay. We co-synthesized KCNB1 and KCNS3, a known heteromeric complex pair, and demonstrated that these channels interact on a liposome. These results indicate that cell-free protein synthesis provides a promising solution for channel studies to overcome the bottleneck of in vitro protein production.

A Low-Cost, Thermostable, Cell-Free Protein Synthesis Platform for On-Demand Production of Conjugate Vaccines.

Cell-free protein synthesis systems that can be lyophilized for long-term, non-refrigerated storage and transportation have the potential to enable decentralized biomanufacturing. However, increased thermostability and decreased reaction cost are necessary for further technology adoption. Here, we identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability and a low-cost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ∼$0.50 per dose after storage at room temperature (∼22 °C) or 37 °C for up to 4 weeks, and ∼$1.00 per dose after storage at 50 °C for up to 4 weeks, with costs based on raw materials purchased at the laboratory scale. We show that these conjugate vaccines generate bactericidal antibodies against enterotoxigenic Escherichia coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our low-cost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.

On-chip microfluidic dual detection of amino acid metabolism disorders using cell-free protein synthesis

Various metabolic diseases are associated with the accumulation of specific amino acids due to abnormal metabolic pathways, and thus can be diagnosed by measuring the level of amino acids in body fluids. However, present methods for amino acid analysis are not readily accessible because they require a complex experimental setup, expensive equipment, and a long processing time. Here, we present a dual sensing microfluidic device that enables fast, portable, and quantitative analysis of target amino acids, harnessing the biological mechanism of protein synthesis. In this device, the working principle of a finger-actuated pumping unit is applied, and the microchannels are designed to perform cell-free synthesis of a reporter protein in response to the target amino acids in the assay samples. Multiple steps required for the translational assay are controlled by the simple operation of two pushbuttons on the device. It is demonstrated that the developed microfluidic device provides precise quantification of two amino acids (methionine and phenylalanine) within 30 min at room temperature. We expect that the application of the presented device can be readily extended to the point-of-care testing of other metabolic compounds.

Ribosome Stalling of N-Linked Glycoproteins in Cell-Free Extracts.

Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free N-glycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype-glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-glycoproteins with advantageous biological and biophysical properties.

Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis

Structural investigations of amyloid fibrils often rely on heterologous bacterial overexpression of the protein of interest. Due to their inherent hydrophobicity and tendency to aggregate as inclusion bodies, many amyloid proteins are challenging to express in bacterial systems. Cell-free protein expression is a promising alternative to classical bacterial expression to produce hydrophobic proteins and introduce NMR-active isotopes that can improve and speed up the NMR analysis. Here we implement the cell-free synthesis of the functional amyloid prion HET-s(218-289). We present an interesting case where HET-s(218-289) directly assembles into infectious fibril in the cell-free expression mixture without the requirement of denaturation procedures and purification. By introducing tailored 13C and 15N isotopes or CF3 and 13CH2F labels at strategic amino-acid positions, we demonstrate that cell-free synthesized amyloid fibrils are readily amenable to high-resolution magic-angle spinning NMR at sub-milligram quantity.

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases.

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal enzymes that catalyze the oxyfunctionalization of non-activated hydrocarbons, making them valuable biocatalysts. Despite the increasing interest in UPOs that has led to the identification of thousands of putative UPO genes, only a few of these have been successfully expressed and characterized. There is currently no universal expression system in place to explore their full potential. Cell-free protein synthesis has proven to be a sophisticated technique for the synthesis of difficult-to-express proteins. In this work, we aimed to establish an insect-based cell-free protein synthesis (CFPS) platform to produce UPOs. CFPS relies on translationally active cell lysates rather than living cells. The system parameters can thus be directly manipulated without having to account for cell viability, thereby making it highly adaptable. The insect-based lysate contains translocationally active, ER-derived vesicles, called microsomes. These microsomes have been shown to allow efficient translocation of proteins into their lumen, promoting post-translational modifications such as disulfide bridge formation and N-glycosylations. In this study the ability of a redox optimized, vesicle-based, eukaryotic CFPS system to synthesize functional UPOs was explored. The influence of different reaction parameters as well as the influence of translocation on enzyme activity was evaluated for a short UPO from Marasmius rotula and a long UPO from Agrocybe aegerita. The capability of the CFPS system described here was demonstrated by the successful synthesis of a novel UPO from Podospora anserina, thus qualifying CFPS as a promising tool for the identification and evaluation of novel UPOs and variants thereof.

Cell-free synthesis and reconstitution of Bax in nanodiscs: Comparison between wild-type Bax and a constitutively active mutant

Bax is a major player in the mitochondrial pathway of apoptosis, by making the Outer Mitochondrial Membrane (OMM) permeable to various apoptogenic factors, including cytochrome c. In order to get further insight into the structure and function of Bax when it is inserted in the OMM, we attempted to reconstitute Bax in nanodiscs. Cell-free protein synthesis in the presence of nanodiscs did not yield Bax-containing nanodiscs, but it provided a simple way to purify full-length Bax without any tag. Purified wild-type Bax (BaxWT) and a constitutively active mutant (BaxP168A) displayed biochemical properties that were in line with previous characterizations following their expression in yeast and human cells followed by their reconstitution into liposomes. Both Bax variants were then reconstituted in nanodiscs. Size exclusion chromatography, dynamic light scattering and transmission electron microscopy showed that nanodiscs formed with BaxP168A were larger than nanodiscs formed with BaxWT. This was consistent with the hypothesis that BaxP168A was reconstituted in nanodiscs as an active oligomer.

Phospholipid synthesis inside phospholipid membrane vesicles

Construction of living artificial cells from genes and molecules can expand our understanding of life system and establish a new aspect of bioengineering. However, growth and division of cell membrane that are basis of cell proliferation are still difficult to reconstruct because a high-yielding phospholipid synthesis system has not been established. Here, we developed a cell-free phospholipid synthesis system that combines fatty acid synthesis and cell-free gene expression system synthesizing acyltransferases. The synthesized fatty acids were sequentially converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of intermediates inhibiting lipid synthesis, sub-millimolar phospholipids could be synthesized within a single reaction mixture. We also performed phospholipid synthesis inside phospholipid membrane vesicles, which encapsulated all the components, and showed the phospholipids localized onto the mother membrane. Our approach would be a platform for the construction of self-reproducing artificial cells since the membrane can grow sustainably.

Potential of mRNA vaccines to become versatile cancer vaccines.

For centuries, therapeutic cancer vaccines have been developed and tried clinically. Way back in the late 19th century, the Father of Immunotherapy, William Coley had discovered that bacterial toxins were effective for inoperable sarcomas. In the 1970s, the Bacillus Calmette-Guérin (BCG) vaccine was repurposed, e.g., for advanced melanomas. Then, therapeutic cancer vaccines based on tumor-associated antigens (found on the surfaces of cancer cells) were tried clinically but apparently have not made a really significant clinical impact. For repurposed pathogen vaccines, only the BCG vaccine was approved in 1989 for local application to treat nonmuscle-invading bladder cancers. Although the mildly toxic vaccine adjuvants deliberately added to conventional pathogen vaccines are appropriate for seasonal applications, when repurposed for continual oncology usage, toxicity may be problematic. In 2010, even with the approval of sipuleucel-T as the very first cancer vaccine (dendritic cell) developed for designated prostate cancers, it has also not made a really significant clinical impact. Perhaps more "user friendly" cancer vaccines should be explored. As from approximately 30 years ago, the safety and effectiveness of mRNA vaccination for oncology had already been studied, the current coronavirus disease 2019 pandemic, though disastrous, has given such progressively advancing technology a kickstart. For oncology, other virtues of mRNA vaccines seem advantageous, e.g., rapid and versatile development, convenient modular design, and entirely cell-free synthesis, are being progressively recognized. Moreover, mRNAs encoding various oncology antigens for vaccination may also be tested with the combi-nation of relatively non-toxic modalities of oncology treatments, e.g., metformin or metronomic (low-dose, prolonged administration) chemotherapy. Admittedly, robust clinical data obtained through good quality clinical trials are mandatory.

Translational Detection of Indole by Complementary Cell-free Protein Synthesis Assay.

The information encoded in a single copy of DNA is processed into a plethora of protein molecules via the cascade of transcription and translation. Thus, the molecular process of gene expression can be considered an efficient biological amplifier from the viewpoint of synthetic biology. Cell-free protein synthesis (CFPS) enables the implementation of this amplification module for in vitro analysis of important biomolecules and avoids many of the problems associated with whole cell-based approaches. Here, we developed a method to analyze indole by using a combination of enzymatic conversion of indole and amino acid-dependent CFPS. In this method, indole molecules in the assay sample are used to generate tryptophan, which is incorporated into signal-generating proteins in the subsequent cell-free synthesis reaction. The activity of cell-free synthesized proteins was successfully used to estimate the indole concentration in the assay sample. In principle, the developed method could be extended to analyses of other important bioactive compounds.

Synthetic biology-powered biosensors based on CRISPR/Cas mediated cascade signal amplification for precise RNA detection

Synthetic biology enabling technologies have been harnessed to create new diagnostic technologies. However, most strategies involve error-prone amplification steps and limitations of accuracy in RNA detection. Here, a cell-free synthetic biology-powered biosensing strategy, termed as SHARK (Synthetic Enzyme Shift RNA Signal Amplifier Related Cas13a Knockdown Reaction), could efficiently and accurately amplify RNA signal by leveraging the collateral cleavage of activated Cas13a to regulate cell-free enzyme synthesis. Based on cascade amplification and tailored enzyme output, SHARK behaves broad compatibility in different scenarios. The portable device based on SHARK was successfully used as SARS-CoV-2 biosensors with high sensitivity and selectivity, and the results were highly consistent with Ct values of qRT-PCR. In addition, when combined with machine learning, SHARK performs bio-computations and thus for cancer diagnosis and staging based on 64 clinical samples. SHARK shows characteristics of precise recognition, cascade amplification and tailored signal outputting comparisons with established assays, presenting significant potential in developing next-generation RNA detection technology.

Purification of therapeutic & prophylactic mRNA by affinity chromatography

In vitro transcribed mRNA is an emerging therapeutic and prophylactic modality with the potential to transform medicine. The drug platform features exceptionally rapid development and versatility of manufacturing processes. Despite the prompt advancement of mRNA from trials to market, purification challenges remain. The cell-free synthesis of mRNA is responsible for the generation of product and process-related impurities, creating the potential for immunogenic effects and decreased translatability into the clinic. Affinity chromatography presents itself as an effective primary capture step for the isolation of functional transcripts from product and some process related impurities. Developing platform processes for the affinity purification of mRNA is hindered by the varying strand lengths of non-amplifying, self-amplifying, and trans-amplifying constructs, with disparities in capacity being observed. Ligand chemistries may contribute to non-specific binding events which remain challenging to characterise. Improved elution and wash conditions may be pursued through novel ligand chemistries, enhanced density and spacing. Regardless of the size or application of the product, the impurities generated by in vitro transcription represent a significant obstacle to the safe administration and long-term storage of mRNA. Affinity chromatography is a valuable tool in overcoming these challenges, with current commercially available products relying heavily on oligo deoxythymidine ligand chemistries. Whilst affinity chromatography is highly valuable in the purification of mRNA, the inability to separate key secondary structures such as double-stranded RNA means it remains to be seen if this technology will adopt the same position as protein A does in mAb manufacture.

Cell-Free Systems Enable the Production of AB5 Toxins for Diagnostic Applications.

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA’s active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin’s mechanism of action.

Phosphorylation of the Hepatitis B Virus Large Envelope Protein.

We here establish the phosphorylation sites in the human hepatitis B virus (HBV) large envelope protein (L). L is involved in several functionally important interactions in the viral life cycle, including with the HBV cellular receptor, HBV capsid, Hsc70 chaperone, and cellular membranes during fusion. We have recently shown that cell-free synthesis of the homologous L protein of duck HBV in wheat germ extract results in very similar phosphorylation events to those previously observed in animal cells. Here, we used mass spectrometry and NMR to establish the phosphorylation patterns of human HBV L protein produced by both in vitro cell-free synthesis and in E. coli with the co-expression of the human MAPK14 kinase. While in the avian virus the phosphorylation of L has been shown to be dispensable for infectivity, the identified locations in the human virus protein, both in the PreS1 and PreS2 domains, raise the intriguing possibility that they might play a functional role, since they are found at strategic sites predicted to be involved in L interactions. This would warrant the further investigation of a possible function in virion formation or cell entry.

Quantitative Analysis of γ-Aminobutyric Acid by Combined Cell-Free Protein Synthesis and Transamination Reactions.

The synthetic power of cells can be harnessed for assaying important analytes, as well as for producing biomolecules. In particular, cell-free protein synthesis (CFPS) can be implemented as a signal amplification module for bioassays, while avoiding many problems associated with whole cell-based microbial biosensors. Here, we developed a method for analyzing γ-aminobutyric acid (GABA) by combining the enzymatic conversion of GABA and amino-acid-dependent CFPS. In this method, GABA molecules in the assay sample are used to generate alanine, which is incorporated into signal-generating proteins in the subsequent cell-free synthesis reaction. The activity of cell-free synthesized proteins was successfully used to estimate the GABA concentration in the assay sample. In principle, the developed method could be extended for the analyses of other important bioactive compounds.

Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display.

Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.

Formation and function of OmpG or OmpA-incorporated liposomes using an in vitro translation system

Outer membrane proteins (OMPs), located on the outer membrane of gram-negative bacteria, have a β-strand structure and form nanopores, which allow passage of ions, sugars, and small molecules. Recently, OMPs have been used as sensing elements to detect biological molecules. OMPs are normally expressed and purified from Escherichia coli (E. coli). Although the cell-free synthesis of OMPs, such as OmpA and OmpG, is achieved in the presence of liposomes and periplasmic chaperones, the amount of OmpA and OmpG incorporated into the nano-sized liposomes is not clear. In this study, after in vitro translation, the incorporation of OmpG into purified nano-sized liposomes with various lipid compositions was investigated. Liposomes containing the synthesized OmpG were purified using a stepwise sucrose density gradient. We report that liposomes prepared with the E. coli lipid extract (PE/PG) had the highest amount of OmpG incorporated compared to liposomes with other lipid compositions, as detected by recording the current across the OmpG containing liposomes using the patch clamp technique. This study reveals some of the requirements for the insertion and refolding of OMPs synthesized by the in vitro translation system into lipid membranes, which plays a role in the biological sensing of various molecules.