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Since its early development and use to decipher the genetic code, in vitro or 'cell-free' systems have been used as an important research tool to understand biochemical mechanisms and metabolic pathways. More recently, due to important engineering advances the technology is rapidly becoming a biomanufacturing platform for protein therapeutics, vaccines, enzyme biocatalysts, fuels, and commodity chemicals. Here we report recent applications and advances in the cell-free biomanufacturing field and the potential of this emerging approach. [ABSTRACT FROM AUTHOR]

Most current vaccine strategies employ live-attenuated or killed versions of the pathogen. While effective in the developed world, expense and cold-chain dependence make broad access to such vaccines difficult in the developing world. Additionally, current vaccine development processes provide only slow responses to emerging or evolved pathogens. Nanoparticle platform technologies for vaccines can overcome these issues. Polymeric nanoparticles have shown pre-clinical success but suffer from structural heterogeneity, instability, and potential off-target immunogenicity issues. Engineered virus-like particles are emerging as a versatile platform for rapid vaccine development. They can be engineered for high stability, facile antigen conjugation in precise orientations, and high potency. Virus-like particle vaccines are poised to deliver the design features required for worldwide distribution of highly effective vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Virus-like particles (VLPs) consist of a virus''s outer shell but without the genome. Similar to the virus, VLPs are monodisperse nano-capsules which have a known morphology, maintain a high degree of symmetry, and can be engineered to encapsidate the desired cargo. VLPs are of great interest for vaccination, drug/gene delivery, imaging, sensing, and material science applications. Here we demonstrate the ability to control the disulfide bond formation in VLPs by directly controlling the redox potential during or after production and assembly of VLPs. The open cell-free protein synthesis environment, which has been reported to produce VLPs at yields comparable or greater than traditional in vivo technologies, was employed. Optimal conditions for disulfide bond formation were found to be VLP dependent, and a cooperative effect in the formation of such bonds was observed. [Copyright &y& Elsevier]

Abstract: Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570±50 μg/mL and 130±30 μg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone. [Copyright &y& Elsevier]

Abstract: Biological technologies for the renewable conversion of biomass to hydrogen, an important chemical feedstock and potential fuel, could reduce greenhouse gas emissions by displacing the current source of virtually all hydrogen: fossil fuels. However, current fermentative methodologies suffer from low productivities and conversion yields. Previous work has shown that purified enzymes from the pentose phosphate pathway can be used to efficiently transfer the reducing equivalents in glucose to NADPH; this approach used a [NiFe] hydrogenase to directly produce hydrogen from NADPH. However, [FeFe] hydrogenases offer much higher hydrogen production activities. We demonstrate a new enzymatic method for the conversion of NADPH reducing equivalents to hydrogen by first using ferredoxin-NADPH-reductase to transfer electrons from NADPH to ferredoxin. The reduced ferredoxin then delivers the electrons to a [FeFe] hydrogenase for hydrogen production. This alternative in vitro pathway enables utilization of the fastest known hydrogenases, [FeFe] hydrogeneases, and activates electron delivery by the native electron donor for these hydrogenases, ferredoxin. We report proof-of-principle data for this synthetic enzyme pathway, showing high volumetric production rates and hydrogenase turnover numbers relative to previous results utilizing [NiFe] hydrogenases. [Copyright &y& Elsevier]

Abstract: [FeFe] hydrogenases catalyze the rapid combination of protons and electrons into hydrogen, but their oxygen sensitivity limits their use in clean renewable hydrogen production applications. The catalytic activity of current [FeFe] hydrogenases is destroyed by oxygen. Here, the discovery of mutant [FeFe] hydrogenases with decreased oxygen sensitivity is described. The new hydrogenases are derived from [FeFe] hydrogenase I of Clostridium pasteurianum (CpI). A cell-free protein synthesis-based screening platform was used to identify an initial mutant from a randomly mutated CpI library. Three mutations were cooperatively responsible for the decreased oxygen sensitivity, and further improvements were identified by saturation mutagenesis at the influential sites. After oxygen exposure under conditions where the enzyme is in the resting state, the mutant hydrogenase retains significantly higher methyl viologen reduction activity than the wild-type enzyme. However, surprisingly, when the enzyme is actively catalyzing hydrogen production during oxygen exposure, the mutant hydrogenase shows no improved oxygen tolerance. This observation highlights the complexity of the oxygen inactivation process in CpI and demonstrates the need to develop a screen that measures hydrogenase oxygen tolerance during catalysis. [Copyright &y& Elsevier]

[FeFe] hydrogenases catalyze rapid H2 production but are highly O2-sensitive. Developing O2-tolerant enzymes is needed for sustainable H2 production technologies, but the lack of a quantitative and predictive assay for O2 tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O2 sensitivity by using an assay employing ferredoxin NADP+ reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H2 production in an O2-containing environment. An alternative assay uses dithionite (DTH) to provide reduced Fd. This second assay measures the remaining hydrogenase activity in periodic samples taken from the NADPH-driven reaction solutions. The second assay validates the more convenient NADPH-driven assay, which better mimics physiological conditions. During development of the NADPH-driven assay and while characterizing the Clostridium pasteurianum (Cp) [FeFe] hydrogenase, CpI, we detected significant rates of direct electron loss from reduced Fd to O2. However, this loss does not interfere with measurement of first order hydrogenase inactivation, providing rate constants insensitive to initial hydrogenase concentration. We show increased activity and O2 tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15-amino acid linker but not for a longer linker. We suggest that this precise, solution phase assay for [FeFe] hydrogenase O2 sensitivity and the insights we provide constitute an important advance toward the discovery of the O2-tolerant [FeFe] hydrogenases required for photosynthetic, biological H2 production. [ABSTRACT FROM AUTHOR]

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. [Display omitted] ● Cell-free protein synthesis has been demonstrated capable of generating numerous product formats suitable as human therapeutics and vaccines, biocatalysts, and sensors. ● Production processes for CFPS machinery and associated reagents have been developed and optimized to support large-scale CFPS applications with viable economics. ● Multiple human clinical studies based on CFPS-derived products are in progress for oncology and infectious disease applications. ● The CFPS approach shows promise as rapid response tools for pandemics and regional disease outbreaks as well as in personalized medicine applications. [ABSTRACT FROM AUTHOR]

Sustainable production of biochemicals from cellulosic biomass is an attractive alternative to chemical production from fossil fuels. We describe the further development of a three protein synthetic pathway to convert NADPH and H + to hydrogen using a ferredoxin-NADP + reductase, a ferredoxin, and the [FeFe] hydrogenase from Clostridium pasteurianum at a rate greater than 14 mmol H 2 L −1 hr −1 using natural enzymes. We also demonstrate the feasibility of coupling this pathway to a cell-free extract to convert glucose to hydrogen in a potentially cost effective manner. Both of these accomplishments serve as the basis for further engineering to optimize both the yield and productivity of a low-cost cell-free process for the production of highly reduced biochemicals. [ABSTRACT FROM AUTHOR]