Cell-Free Display Techniques for Protein Evolution.

Cell-free protein synthesis (CFPS) with flexibility and controllability can provide a powerful platform for high-throughput screening of biomolecules, especially in the evolution of peptides or proteins. In this chapter, the emerging strategies for enhancing the protein expression level using different source strains, energy systems, and template designs in constructing CFPS systems are summarized and discussed in detail. In addition, we provide an overview of the ribosome display, mRNA display, cDNA display, and CIS display in vitro display technologies, which can couple genotype and phenotype by forming fusion complexes. Moreover, we point out the trend that improving the protein yields of CFPS itself can offer more favorable conditions for maintaining library diversity and display efficiency. It is hoped that the novel CFPS system can accelerate the development of protein evolution in biotechnological and medical applications.

Membrane protein synthesis: no cells required.

Despite advances in membrane protein (MP) structural biology and a growing interest in their applications, these proteins remain challenging to study. Progress has been hindered by the complex nature of MPs and innovative methods will be required to circumvent technical hurdles. Cell-free protein synthesis (CFPS) is a burgeoning technique for synthesizing MPs directly into a membrane environment using reconstituted components of the cellular transcription and translation machinery in vitro. We provide an overview of CFPS and how this technique can be applied to the synthesis and study of MPs. We highlight numerous strategies including synthesis methods and folding environments, each with advantages and limitations, to provide a survey of how CFPS techniques can advance the study of MPs.

Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System.

Cell-free protein synthesis (CFPS) systems are emerging as powerful platforms for in vitro protein production, which leads to the development of new CFPS systems for different applications. To expand the current CFPS toolkit, here we develop a novel CFPS system derived from a chassis microorganism Klebsiella pneumoniae, an important industrial host for heterologous protein expression and the production of many useful chemicals. First, we engineered the K. pneumoniae strain by deleting a capsule formation-associated wzy gene. This capsule-deficient strain enabled easy collection of the cell biomass for preparing cell extracts. Then, we optimized the procedure of cell extract preparation and the reaction conditions for CFPS. Finally, the optimized CFPS system was able to synthesize a reporter protein (superfolder green fluorescent protein, sfGFP) with a maximum yield of 253 ± 15.79 µg/mL. Looking forward, our K. pneumoniae-based CFPS system will not only expand the toolkit for protein synthesis, but also provide a new platform for constructing in vitro metabolic pathways for the synthesis of high-value chemicals.

Cell-free technologies for biopharmaceutical research and production.

Cell-free protein synthesis (CFPS) technologies have grown from lab-scale research tools to biopharmaceutical production at the Good Manufacturing Practice manufacturing scale. Multiple human clinical trials are in progress with CFPS-based products. In addition, applications of CFPS in research have continued to expand over the years and play an important role in biopharmaceutical product discovery and development. The unique, open nature of CFPS has enabled efficient non-natural amino acid (nnAA) incorporation into protein products, which expands the range of biotherapeutics that can be considered for novel treatments. The flexibility and speed of CFPS combined with novel nnAA capabilities are poised to open a new chapter in the continuing evolution of biotherapies.

A cell-free approach to identify binding hotspots in plant immune receptors.

Plant immune receptors are often difficult to express heterologously, hindering study of direct interactions between these receptors and their targets with traditional biochemical approaches. The cell-free method ribosome display (RD) enables expression of such recalcitrant proteins by keeping each nascent polypeptide chain tethered to its ribosome, which can enhance protein folding by virtue of its size and solubility. Moreover, in contrast to an in planta readout of receptor activity such as a hypersensitive response that conflates binding and signaling, RD enables direct probing of the interaction between plant immune receptors and their targets. Here, we demonstrate the utility of this approach using tomato recognition of Trichoderma viride ethylene-inducing xylanase (EIX) as a case study. Leveraging the modular nature of the tomato LeEIX2 and LeEIX1 leucine-rich repeat (LRR) receptors, we applied an entropy-informed algorithm to maximize the information content in our receptor segmentation RD experiments to identify segments implicated in EIX binding. Unexpectedly, two distinct EIX-binding hotspots were discovered on LeEIX2 and both hotspots are shared with decoy LeEIX1, suggesting that their contrasting receptor functions are not due to differential modes of ligand binding. Given that most plant immune receptors are thought to engage targets via their LRR sequences, this approach should be of broad utility in rapidly identifying their binding hotspots.

Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides.

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis. SIGNIFICANCE: The developed method can be applied for the preparation of internal standard peptides without limiting the number of peptides to be synthesized, which may result in more practical screening of quantitatively reliable peptides, one of the fundamental steps in the reliable absolute quantification using MS. Furthermore, the method is highly versatile for proteome analysis of any organisms or species without any cDNA or SIL peptide libraries. The quantification can be finished in a few days including design and preparation of appropriate SIL peptides using small-scale batch cell-free reactions, which has a potential to be a part of the standard methodology in a field of quantitative proteomics.

Synthesis of Tyrosol and Hydroxytyrosol Glycofuranosides and Their Biochemical and Biological Activities in Cell-Free and Cellular Assays.

Tyrosol (T) and hydroxytyrosol (HOT) and their glycosides are promising candidates for applications in functional food products or in complementary therapy. A series of phenylethanoid glycofuranosides (PEGFs) were synthesized to compare some of their biochemical and biological activities with T and HOT. The optimization of glycosylation promoted by environmentally benign basic zinc carbonate was performed to prepare HOT α-L-arabino-, ß-D-apio-, and ß-D-ribofuranosides. T and HOT ß-D-fructofuranosides, prepared by enzymatic transfructosylation of T and HOT, were also included in the comparative study. The antioxidant capacity and DNA-protective potential of T, HOT, and PEGFs on plasmid DNA were determined using cell-free assays. The DNA-damaging potential of the studied compounds for human hepatoma HepG2 cells and their DNA-protective potential on HepG2 cells against hydrogen peroxide were evaluated using the comet assay. Experiments revealed a spectrum of different activities of the studied compounds. HOT and HOT ß-D-fructofuranoside appear to be the best-performing scavengers and protectants of plasmid DNA and HepG2 cells. T and T ß-D-fructofuranoside display almost zero or low scavenging/antioxidant activity and protective effects on plasmid DNA or HepG2 cells. The results imply that especially HOT ß-D-fructofuranoside and ß-D-apiofuranoside could be considered as prospective molecules for the subsequent design of supplements with potential in food and health protection.

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts.

Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing.

Characterizing and Improving Reaction Times for E. coli-Based Cell-Free Protein Synthesis.

Cell-free protein synthesis (CFPS) is a platform biotechnology that has enabled the on-demand synthesis of proteins for a variety of applications. Numerous advances have improved the productivity of the CFPS platform to result in high-yielding reactions; however, many applications remain limited due to long reaction times. To overcome this limitation, we first established the benchmarks reaction times for CFPS across in-house E. coli extracts and commercial kits. We then set out to fine-tune our in-house extract systems to improve reaction times. Through the optimization of reaction composition and titration of low-cost additives, we have identified formulations that reduce reaction times by 30-50% to obtain high protein titers for biomanufacturing applications, and reduce times by more than 50% to reach the sfGFP detection limit for applications in education and diagnostics. Under optimum conditions, we report the visible observation of sfGFP signal in less than 10 min. Altogether, these advances enhance the utility of CFPS as a rapid, user-defined platform.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System.

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal.

The field of liquid biopsy has seen extensive growth in recent decades, making it one of the most promising areas in molecular diagnostics. Circulating cell-free DNA (ccfDNA) especially is used as an analyte in a growing number of diagnostic assays. These assays require specified preanalytical workflows delivering ccfDNA in qualities and quantities that facilitate correct and reliable results. As each step and component used in the preanalytical process has the potential to influence the assay sensitivity and other performance characteristics, it is key to find an unbiased experimental setup to test these factors in diagnostic or research laboratories. We defined one such setup by using blood from healthy subjects and commercially available products for blood collection, spike-in material, ccfDNA isolation, and qPCR assays. As the primary read-out, we calculated the probit model-based LOD95 (limit of detection of the 95th percentile) from the qPCR assay results. In a proof of principle study we tested two different but widely used blood ccfDNA profile stabilization technologies in blood collection tubes, the Cell-Free DNA BCT and the PAXgene Blood ccfDNA Tube. We tested assays for three different EGFR gene mutations and one BRAF gene mutation. The study design revealed differences in performance between the two tested technologies for all four mutations. In conclusion, we successfully established a blueprint for a test procedure capable of verifying and validating a liquid biopsy workflow from blood collection to the analytical result.

DNA Damage Response in Xenopus laevis Cell-Free Extracts.

The DNA damage response (DDR) is a coordinated cellular response to a variety of insults to the genome. DDR initiates the activation of cell cycle checkpoints preventing the propagation of damaged DNA followed by DNA repair, which are both critical in maintaining genome integrity. Several model systems have been developed to study the mechanisms and complexity of checkpoint function. Here we describe the application of cell-free extracts derived from Xenopus eggs as a model system to investigate signaling from DNA damage, modulation of DNA replication, checkpoint activation, and ultimately DNA repair. We outline the preparation of cell-free extracts, DNA substrates, and their subsequent use in assays aimed at understanding the cellular response to DNA damage. Cell-free extracts derived from the eggs of Xenopus laevis remain a robust and versatile system to decipher the biochemical steps underlying this essential characteristic of all cells, critical for genome stability.

A Method for Determining the Kinetics of Small-Molecule-Induced Ubiquitination.

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

Human blood contains circulating cell-free mitochondria, but are they really functional?

Dache et al. (FASEB J 34: 3616-3630, 2020) recently reported the presence of respiratory-competent cell-free mitochondria in human blood (up to 3.7 × 106 per mL of blood), providing exciting perspectives on the potential role of these extracellular mitochondria. Although their evidence for the presence of cell-free mitochondria in human blood is compelling, their conclusion that these cell-free mitochondria are respiratory competent or functional has to be reevaluated. To this end, we evaluated the functionality of cell-free mitochondria in human blood using high-resolution respirometry and mitochondria extracted from platelets of the same blood samples as positive controls. Although cell-free mitochondria were present in human plasma (i.e., significant MitoTracker Green fluorescence and complex IV activity), there was no evidence suggesting that their mitochondrial electron transport system (ETS) was functional (i.e., respiration rate not significantly different from 0; no significant responses to ADP, uncoupler, or mitochondrial inhibitors oligomycin and antimycin A). Yet, in vitro complex IV activity was detectable and even slightly higher than levels found in mitochondria extracted from platelets, suggesting that cell-free mitochondria in human blood are likely to only retain a nonfunctional part of the ETS. Despite being unlikely to be fully functional in the narrow sense (i.e., capable of oxidative phosphorylation), circulating cell-free mitochondria may have significant physiological roles that remain to be elucidated.NEW & NOTEWORTHY The recently reported cell-free mitochondria in human blood have been thought to be respiratory competent, giving rise to speculation about their biological function(s). By characterizing their bioenergetics in vitro, we show that circulating cell-free mitochondria are unlikely to be functional in vivo since they display no potential for oxidative phosphorylation.

In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins.

Efforts to expand the scope of ribosome-mediated polymerization to incorporate noncanonical amino acids (ncAAs) into peptides and proteins hold promise for creating new classes of enzymes, therapeutics, and materials. Recently, the integrated synthesis, assembly, and translation (iSAT) system was established to construct functional ribosomes in cell-free systems. However, the iSAT system has not been shown to be compatible with genetic code expansion. Here, to address this gap, we develop an iSAT platform capable of manufacturing pure proteins with site-specifically incorporated ncAAs. We first establish an iSAT platform based on extracts from genomically recoded Escherichia coli lacking release factor 1 (RF-1). This permits complete reassignment of the amber codon translation function. Next, we optimize orthogonal translation system components to demonstrate the benefits of genomic RF-1 deletion on incorporation of ncAAs into proteins. Using our optimized platform, we demonstrate high-level, multi-site incorporation of p-acetyl-phenylalanine (pAcF) and p-azido-phenylalanine into superfolder green fluorescent protein (sfGFP). Mass spectrometry analysis confirms the high accuracy of incorporation for pAcF at one, two, and five amber sites in sfGFP. The iSAT system updated for ncAA incorporation sets the stage for investigating ribosomal mutations to better understand the fundamental basis of protein synthesis, manufacturing proteins with new properties, and engineering ribosomes for novel polymerization chemistries.

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.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.

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 membrane proteins 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 468 kDa TET2 protein assembly for NMR investigations is presented.

Methods for Expression of Recombinant Proteins Using a Pichia pastoris Cell-Free System.

Cell-free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell-free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell-free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post-translational modifications. Here, we describe methods for conducting cell-free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription-translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike. © 2020 The Authors. Basic Protocol 1: Preparation of Pichia pastoris cell lysate Basic Protocol 2: Coupled in vitro transcription and translation Basic Protocol 3: Determining luciferase production from cell-free protein synthesis reactions Alternate Protocol 1: Linked in vitro transcription and translation Alternate Protocol 2: Quantifying HSA protein concentration Support Protocol 1: Preparation of mRNA by in vitro transcription for linked transcription and translation.

A bio-inspired cell-free system for cannabinoid production from inexpensive inputs.

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l-1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.