Impact of Chemical Dynamics of Commercial PURE Systems on Malachite Green Aptamer Fluorescence.

The malachite green aptamer (MGapt) is known for its utility in RNA measurement in vivo and in lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect RNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence, which leads to inaccurate RNA calculations. Specific to the commercial system, we posit that MGapt fluorescence is significantly affected by the system's chemical properties, governed notably by the presence of dithiothreitol (DTT). We propose a model that, on average, accurately predicts MGapt measurement within a 10% margin, leveraging DTT concentration as a critical factor. This model sheds light on the complex dynamics of MGapt in cell-free systems and underscores the importance of considering environmental factors in RNA measurements using aptamers.

Towards Self-regeneration: Exploring the Limits of Protein Synthesis in the Protein Synthesis Using Recombinant Elements (PURE) Cell-free Transcription-Translation System.

Self-regeneration is a key function of living systems that needs to be recapitulated in vitro to create a living synthetic cell. A major limiting factor for protein self-regeneration in the PURE cell-free transcription-translation system is its high protein concentration, which far exceeds the system's protein synthesis rate. Here, we were able to drastically reduce the nonribosomal PURE protein concentration up to 97.3% while increasing protein synthesis efficiency. Although crowding agents were not effective in the original PURE formulation, we found that in highly dilute PURE formulations, addition of 6% dextran considerably increased protein synthesis rate and total protein yield. These new PURE formulations will be useful for many cell-free synthetic biology applications, and we estimate that PURE can now support the complete self-regeneration of all 36 nonribosomal proteins, which is a critical step toward the development of a universal biochemical constructor and living synthetic cell.

Reconstituted Cell-free Translation Systems for Exploring Protein Folding and Aggregation.

Protein folding is crucial for achieving functional three-dimensional structures. However, the process is often hampered by aggregate formation, necessitating the presence of chaperones and quality control systems within the cell to maintain protein homeostasis. Despite a long history of folding studies involving the denaturation and subsequent refolding of translation-completed purified proteins, numerous facets of cotranslational folding, wherein nascent polypeptides are synthesized by ribosomes and folded during translation, remain unexplored. Cell-free protein synthesis (CFPS) systems are invaluable tools for studying cotranslational folding, offering a platform not only for elucidating mechanisms but also for large-scale analyses to identify aggregation-prone proteins. This review provides an overview of the extensive use of CFPS in folding studies to date. In particular, we discuss a comprehensive aggregation formation assay of thousands of Escherichia coli proteins conducted under chaperone-free conditions using a reconstituted translation system, along with its derived studies.

N-Terminal Amino Acid Affects the Translation Efficiency at Lower Temperatures in a Reconstituted Protein Synthesis System.

The Escherichia coli (E. coli)-based protein synthesis using recombinant elements (PURE) system is a cell-free protein synthesis system reconstituted from purified factors essential for E. coli translation. The PURE system is widely used for basic and synthetic biology applications. One of the major challenges associated with the PURE system is that the protein yield of the system varies depending on the protein. Studies have reported that the efficiency of translation is significantly affected by nucleotide and amino acid sequences, especially in the N-terminal region. Here, we investigated the inherent effect of various N-terminal sequences on protein synthesis using the PURE system. We found that a single amino acid substitution in the N-terminal region significantly altered translation efficiency in the PURE system, especially at low temperatures. This result gives us useful suggestions for the expression of the protein of interest in vitro and in vivo.

Changes in Coding and Efficiency through Modular Modifications to a One Pot PURE System for In Vitro Transcription and Translation.

The incorporation of unnatural amino acids is an attractive method for improving or bringing new and novel functions in peptides and proteins. Cell-free protein synthesis using the Protein Synthesis Using Recombinant Elements (PURE) system is an attractive platform for efficient unnatural amino acid incorporation. In this work, we further adapted and modified the One Pot PURE to obtain a robust and modular system for enzymatic single-site-specific incorporation of an unnatural amino acid. We demonstrated the flexibility of this system through the introduction of two different orthogonal aminoacyl tRNA synthetase:tRNA pairs that suppressed two distinctive stop codons in separate reaction mixtures.

Regulated N-Terminal Modification of Proteins Synthesized Using a Reconstituted Cell-Free Protein Synthesis System.

The N-terminal modification of nascent proteins, such as acetylation and myristoylation, is one of the most abundant post-translational modifications. To analyze the function of the modification, it is important to compare the modified and unmodified proteins under defined conditions. However, it is technically difficult to prepare unmodified proteins because cell-based systems contain endogenous modification systems. In this study, we developed a cell-free method to conduct N-terminal acetylation and myristoylation of nascent proteins in vitro using a reconstituted cell-free protein synthesis system (PURE system). Proteins synthesized using the PURE system were successfully acetylated or myristoylated in a single-cell-free mixture in the presence of modifying enzymes. Furthermore, we performed protein myristoylation in giant vesicles, which resulted in their partial localization to the membrane. Our PURE-system-based strategy is useful for the controlled synthesis of post-translationally modified proteins.

DNA-Encoded Immunoassay in Picoliter Drops: A Minimal Cell-Free Approach.

Immunoassays have emerged as indispensable bioanalytical tools but necessitate long preliminary steps for the selection, production, and purification of the antibody(ies) to be used. Here is explored the paradigm shift of creating a rapid and purification-free assay in picoliter drops where the antibody is expressed from coding DNA and its binding to antigens concomitantly characterized in situ. Efficient synthesis in bulk of various functional variable domains of heavy-chain only antibodies (VHH) using reconstituted cell-free expression media, including an anti-green fluorescent protein VHH, is shown first. A microfluidic device is then used to generate monodisperse drops (30 pL) containing all the assay components, including a capture scaffold, onto which the accumulation of VHH:antigen produces a specific fluorescent signal. This allows to assess, in parallel or sequentially at high throughput (500 Hz), the VHH-antigen binding and its specificity in less than 3 h, directly from a VHH-coding DNA, for multiple VHH sequences, various antigens and down to DNA concentrations as low as 12 plasmids per drop. It is anticipated that the ultraminiaturized format, robustness, and programmability of this novel cell-free immunoassay concept will constitute valuable assets in fields as diverse as antibody discovery, point-of-care diagnostics, synthetic biology, and/or bioanalytical assays.

Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein-Protein Interaction between CTLA-4 and B7-1.

Currently, various pharmaceutical modalities are being developed rapidly. Targeting protein-protein interactions (PPIs) is an important objective in such development. Cyclic peptides, because they have good specificity and activity, have been attracting much attention as an alternative to antibody drugs. However, cyclic peptides involve some difficulties, such as oral availability and cell permeability. Therefore, while small-molecule drugs still present many benefits, the screening of functional small-molecule compounds targeting PPIs requires a great deal of time and effort, including structural analysis of targets and hits. In this study, we investigated a rational two-step strategy to design small-molecule compounds targeting PPIs. First, we obtained inhibitory cyclic peptides that bind to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) by ribosomal display using PUREfrex® (PUREfrex®RD) to get structure-activity relation (SAR) information. Based on that information, we converted cyclic peptides to small molecules using PepMetics® scaffolds that can mimic the α-helix or ß-turn of the peptide. Finally, we succeeded in generating small-molecule compounds with good IC50 (single-digit µM values) against CTLA-4. This strategy is expected to be a useful approach for small-molecule design targeting PPIs, even without having structural information such as that associated with X-ray crystal structures.

Transfer RNA Synthesis-Coupled Translation and DNA Replication in a Reconstituted Transcription/Translation System.

Transfer RNAs (tRNAs) are key molecules involved in translation. In vitro synthesis of tRNAs and their coupled translation are important challenges in the construction of a self-regenerative molecular system. Here, we first purified EF-Tu and ribosome components in a reconstituted translation system of Escherichia coli to remove residual tRNAs. Next, we expressed 15 types of tRNAs in the repurified translation system and performed translation of the reporter luciferase gene depending on the expression. Furthermore, we demonstrated DNA replication through expression of a tRNA encoded by DNA, mimicking information processing within the cell. Our findings highlight the feasibility of an in vitro self-reproductive system, in which tRNAs can be synthesized from replicating DNA.

Cell-Free Mutant Analysis Combined with Structure Prediction of a Lasso Peptide Biosynthetic Protease B2.

Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble, and no experimentally solved structures are available. Here, we performed a rapid semicomprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (FusB2), using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 FusB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, FusB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure-function research of proteins for which no structures are available.

Discovery of IL-5-binding unnatural cyclic peptides from multiple libraries by directed evolution.

Interleukin-5 (IL-5) is a type 2 cytokine involved in various allergic diseases, including severe eosinophilic asthma. In this study, we performed directed evolution against human IL-5 using systematic evolution of ligands by exponential enrichment (SELEX) from multiple mRNA-displayed peptide libraries. Peptide libraries were prepared with Escherichia coli-based reconstituted cell-free transcription and translation coupling system (PURE system) and spontaneously cyclized using multiple intramolecularly thiol-reactive benzoic acid-derived linkers, which were ribosomally incorporated through genetic code expansion. We successfully identified multiple novel IL-5-binding unnatural cyclic peptides with different cyclization linkers from multiple highly diverse mRNA-displayed libraries. Chemical dimerization was also performed to increase the avidity of unnatural cyclic IL-5-binding peptides. The novel IL-5-binding unnatural cyclic peptides discovered in this study could be used in various research, therapeutic, and diagnostic applications involving IL-5 signaling.

Ribosomal Protein S1 Improves the Protein Yield of an In Vitro Reconstituted Cell-Free Translation System.

Cell-free expression systems, such as the highly purified in vitro reconstituted PURExpress, hold great promise for engineering biological and life-similar systems by exploiting the ability to perform transcription and translation (TX-TL) outside the constraints of living cells, including for example the expression of recombinant proteins that are difficult or toxic to produce in vivo. Currently, the scope of applications utilizing purified reconstituted TX-TL systems is challenged by poor system performance resulting from limitations in the ribosome and ribosome-associated processes, leading to low protein yields. Because of the transient nature of ribosomal protein S1's interaction with the ribosome, the ribosomes in a reconstituted translation system contain varying amounts of S1, potentially impacting translation initiation and the recruitment of mRNA to the 30S ribosomal subunit. Here we report that by being supplemented with purified recombinant S1 the protein yields can be doubled when using a commercial in vitro reconstituted TX-TL system. We hypothesize that the addition of S1 increases the fraction of functional ribosomes available in the in vitro reaction. Improved yields are shown for different reporter proteins (EYFP, sfGFP, and mRFP) and in different 5'UTR contexts (strong, medium, and weak ribosome binding site), including the expression of a highly structured RNA (PSIV IRES). Overall, fine-tuning the S1 concentration provides a previously overlooked venue to increase protein yield by targeting ribosome composition and translation initiation.

Efficient and Precise Protein Synthesis in a Cell-Free System Using a Set of In Vitro Transcribed tRNAs with Nucleotide Modifications.

Reconstitution of a complicated system with a minimal set of components is essential for understanding the mechanisms of how the input is reflected in the output, which is fundamental for further engineering of the corresponding system. We have recently developed a reconstituted cell-free protein synthesis system equipped only with 21 in vitro transcribed tRNAs, one of the minimal systems for understanding the genetic code decoding mechanisms. Introduction of several nucleotide modifications to the transcribed tRNAs showed improvement of both protein synthesis efficiency and its fidelity, suggesting various combinations of tRNAs and their modifications can be evaluated in the developed system. In this chapter, we describe how to prepare this minimal system. Methods for preparing the transcribed tRNAs, their modifications, and the protein production using the set of prepared tRNAs are shown.

In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins.

Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.

Assessment of Colorimetric Reporter Enzymes in the PURE System.

Colorimetric reporter enzymes are useful for generating eye-readable biosensor readouts that do not require a device to interpret, an attractive property for applications in remote or developing parts of the world. The use of cell-free gene expression further facilitates such applications via amenability to lyophilization and incorporation into materials like paper. Currently, detection of multiple analytes simultaneously with these systems requires multiple reactions or a device. Here we evaluate seven enzymes and 15 corresponding substrates for functionality in a particular cell-free expression system known as PURE. We report eight enzyme/substrate pairs spanning four enzymes that are compatible with PURE. Of the four enzymes, three pairings exhibit no cross-reactivity. We finally show that at least one pairing can be used to create a third color when both are present, highlighting the potential use of these reporters for multiplex sensing.

PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high-throughput sequencing.

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE-based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

Directed evolution of dibenzocyclooctyne-reactive peptide tags for protein labeling.

Protein labeling with a functional molecule is a technique widely used for protein research. The covalent reaction of self-labeling peptide tags with synthetic probe-modified small molecules enables tag-fused protein labeling with chemically diverse molecules, including fluorescent probes. We report the discovery, by in vitro directed evolution, of a novel 23-mer dibenzocyclooctyne (DBCO)-reactive peptide (DRP) tag using Systematic Evolution of Ligands by EXponential enrichment (SELEX) with a combination of a reconstituted cell-free translation system (PURE system) and cDNA display. The N- and C-terminal DRP truncations created a shorter 16-mer DBCO-reactive peptide (sDRP) tag without significant reactivity reduction. By fusing the sDRP tag to a model protein, we showed the chemical labeling and in-gel fluorescence imaging of the sDRP-fused protein using a fluorescent DBCO probe. Results showed that sDRP tag-mediated protein labeling has potential for use as a basic molecular tool in a variety of applications for protein research.

In vitro display evolution of the PURE system-expressed TNFα-binding unnatural cyclic peptide containing an N-methyl-d-amino acid.

Tumor necrosis factor-alpha (TNFα) is a multifunctional cytokine associated with inflammation, immune responses, and autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. In the present study, we performed in vitro selection, systematic evolution of ligands by exponential enrichment (SELEX) against human TNFα from mRNA-displayed peptide library prepared with Escherichia coli-reconstituted cell-free transcription/translation system (PURE system) and cyclized by N-chloroacetyl-N-methyl-d-phenylalanine incorporated by genetic code expansion (sense suppression). We identified a novel TNFα-binding thioether-cyclized peptide that contains an N-methyl-d-phenylalanine. Since cyclic structure and presence of an N-methyl-d-amino acid can increase proteolytic stability, the TNFα binding peptide has potential to be used for therapeutic, research and diagnostic applications.

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.

The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins.

Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from Escherichia coli cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into E. coli membrane vesicles. MPIase-depleted membrane vesicles lacked both 3L-Pf3 coat and MtlA integration, indicating that MPIase is involved in the integration of both proteins. We developed a reconstitution system in which disordered spontaneous integration was precluded, which revealed that SecYEG, YidC, or both, are not sufficient for Sec-dependent and -independent integration. Although YidC had no effect on MPIase-dependent integration of Sec-independent substrates in the conventional assay system, YidC significantly accelerated the integration when the substrate amounts were increased in our PURE system-based assay. Similar acceleration by YidC was observed for MtlA integration. YidC mutants with amino acid substitutions in the hydrophilic cavity inside the membrane were defective in the acceleration of the Sec-independent integration. Of note, MPIase was up-regulated upon YidC depletion. These results indicate that YidC accelerates the MPIase-dependent integration of membrane proteins, suggesting that MPIase and YidC function sequentially and cooperatively during the catalytic cycle of membrane protein integration.