Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system.

Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis. Cell-free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell-free systems make them compelling candidates for future biotherapeutic screening and production.

Creating a completely "cell-free" system for protein synthesis.

Cell-free protein synthesis is a promising tool to take biotechnology outside of the cell. A cell-free approach provides distinct advantages over in vivo systems including open access to the reaction environment and direct control over all chemical components for facile optimization and synthetic biology integration. Promising applications of cell-free systems include portable diagnostics, biotherapeutics expression, rational protein engineering, and biocatalyst production. The highest yielding and most economical cell-free systems use an extract composed of the soluble component of lysed Escherichia coli. Although E. coli lysis can be highly efficient (>99.999%), one persistent challenge is that the extract remains contaminated with up to millions of cells per mL. In this work, we examine the potential of multiple decontamination strategies to further reduce or eliminate bacteria in cell-free systems. Two strategies, sterile filtration and lyophilization, effectively eliminate contaminating cells while maintaining the systems' protein synthesis capabilities. Lyophilization provides the additional benefit of long-term stability at storage above freezing. Technologies for personalized, portable medicine and diagnostics can be expanded based on these foundational sterilized and completely "cell-free" systems.

The emerging age of cell-free synthetic biology.

The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology.

Lyophilized Escherichia coli-based cell-free systems for robust, high-density, long-term storage.

Cell-free protein synthesis (CFPS) is a versatile tool for rapid recombinant protein production and engineering. One drawback of cell-free technology is the necessity to store the major components-cell extracts and energy systems-below freezing in bulky aqueous solutions. Here we describe simple methods for lyophilizing extracts and preparing powdered energy systems for CFPS. These techniques allow for high-density storage of cell-free systems that are more robust against temperature and bacterial degradation. Our methods have the potential to decrease storage expenses, allow for longer shelf-life of cell extracts at room temperature, and enable durable portable protein production technologies.

Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates.

Site-specific incorporation of unnatural amino acids (uAAs) during protein synthesis expands the proteomic code through the addition of unique residue chemistry. This field provides a unique tool to improve pharmacokinetics, cancer treatments, vaccine development, proteomics and protein engineering. The limited ability to predict the characteristics of proteins with uAA-incorporation creates a need for a low-cost system with the potential for rapid screening. Escherichia coli-based cell-free protein synthesis is a compelling platform for uAA incorporation due to the open and accessible nature of the reaction environment. However, typical cell-free systems can be expensive due to the high cost of energizing reagents. By employing alternative energy sources, we reduce the cost of uAA-incorporation in CFPS by 55%. While alternative energy systems reduce cost, the time investment to develop gene libraries can remain cumbersome. Cell-free systems allow the direct use of PCR products known as linear expression templates, thus alleviating tedious plasmid library preparations steps. We report the specific costs of CFPS with uAA incorporation, demonstrate that LETs are suitable expression templates with uAA-incorporation, and consider the substantial reduction in labor intensity using LET-based expression for CFPS uAA incorporation.

Reengineering viruses and virus-like particles through chemical functionalization strategies.

Increasing demands from nanotechnology require increasingly more rigorous methods to control nanoparticle traits such as assembly, size, morphology, monodispersity, stability, and reactivity. Viruses are a compelling starting point for engineering nanoparticles, as eons of natural biological evolution have instilled diverse and desirable traits. The next step is to reengineer these viruses into something functional and useful. These reengineered particles, or virus-based nanoparticles (VNPs), are the foundation for many promising new technologies in drug delivery, targeted delivery, vaccines, imaging, and biocatalysis. To achieve these end goals, VNPs must often be manipulated genetically and post-translationally. We review prevailing strategies of genetic and noncovalent functionalization and focus on the covalent modifications using natural and unnatural amino acid residues.