New frontiers and applications of cell-free systems.

Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation of synthetic proteins with desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment and in vivo imaging. The positioning of the ncAA-responsive codon within the protein’s coding sequence is critical in order to maintain protein function, achieve high yields of ncAA-containing protein, and allow effective conjugation. Cell-free ncAA incorporation is of particular interest due to the open nature of cell-free systems and their concurrent ease of manipulation. In this study, we report a straightforward workflow to inquire ncAA positions in regard to incorporation efficiency and protein functionality in a Chinese hamster ovary (CHO) cell-free system. As a model, the well-established orthogonal translation components Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and tRNATyrCUA were used to site-specifically incorporate the ncAA p-azido-l-phenylalanine (AzF) in response to UAG codons. A total of seven ncAA sites within an anti-epidermal growth factor receptor (EGFR) single-chain variable fragment (scFv) N-terminally fused to the red fluorescent protein mRFP1 and C-terminally fused to the green fluorescent protein sfGFP were investigated for ncAA incorporation efficiency and impact on antigen binding. The characterized cell-free dual fluorescence reporter system allows screening for ncAA incorporation sites with high incorporation efficiency that maintain protein activity. It is parallelizable, scalable, and easy to operate. We propose that the established CHO-based cell-free dual fluorescence reporter system can be of particular interest for the development of antibody-drug conjugates (ADCs).

Rewiring translation – Genetic code expansion and its applications

The paper is devoted to the study of the legal regulation of public relations in the field of synthetic biology. The purpose of the paper is to identify the key features of the concept of «synthetic biology»; to conduct a study of regulatory legal acts at the global level, the level of interstate integration associations, and the Russian Federation, which are devoted to regulation in the field of synthetic biology; to identify the main aspects and directions in which the concept of «synthetic biology» is used; to formulate proposals for the legal regulation of relevant relations at the at the level of the Russian Federation. The various definitions of the concept of «synthetic biology» in Russian and foreign doctrinal sources, as well as in international legal documents and documents of international organizations are analyzed. The analysis of the statutory regulation of public relations in the field of synthetic biology is carried out. The research utilizes dogmatic and formal logical methods, an axiological approach and a method of comparison. Based on the results of the analysis, the essential features of the concept of «synthetic biology» are identified, conclusions are drawn about the main aspects and directions in which this concept is used. Proposals have been formulated for the legal regulation of relevant relations at the level of the Russian Federation, according to which it is advisable to develop regulation of the use of synthetic biology technologies for the possible creation of pathogenic organisms and other threats in the field of biosafety, while regulating the use of synthetic biology technologies in other areas is impractical, since the relevant public relations fall under existing regulation in more general areas — regulation of scientific research and regulation in the field of biotechnology, bioeconomics, and genetic technologies.

An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins

Cell-free protein synthesis (CFPS) has emerged as a novel protein expression platform. Especially the incorporation of non-canonical amino acids (ncAAs) has led to the development of numerous flexible methods for efficient and extensive expression of artificial proteins. Approaches were developed to eliminate the endogenous competition for ncAAs and engineer translation factors, which significantly enhanced the incorporation efficiency. Furthermore, in vitro aminoacylation methods can be conveniently combined with cell-free systems, extensively expanding the available ncAAs with novel and unique moieties. In this review, we summarize the recent progresses on the efficient and extensive incorporation of ncAAs by different strategies based on the elimination of competition by endogenous factors, translation factors engineering and extensive incorporation of novel ncAAs coupled with in vitro aminoacylation methods in CFPS. We also aim to offer new ideas to researchers working on ncAA incorporation techniques in CFPS and applications in various emerging fields.

Open cell-free translation systems based on Escherichia coli cell lysates have successfully been used to produce antibodies and antibody fragments. In this study, we demonstrate the cell-free expression of functional single-chain antibody variable fragments (scFvs) in a eukaryotic and endotoxin-free in vitro translation system based on Spodoptera frugiperda (Sf21) insect cell extracts. Three scFv candidates with different specificities were chosen as models. The first scFv candidate SH527-IIA4 specifically discriminates between its phosphorylated (SMAD2-P) and nonphosphorylated antigens (SMAD2) (where SMAD is mothers against decapentaplegic homolog 2), whereas the second scFv candidate SH527-IIC10 recognizes both, SMAD2-P and SMAD2. The third scFv candidate SH855-C11 binds specifically to a linear epitope of the CXC chemokine receptor type 5. The translocation of antibody fragments into the lumen of endogenous microsomal vesicles, which are contained in the lysate, was facilitated by fusion of scFv genes to the insect cell specific signal sequence of honeybee melittin. We compared the binding capabilities of scFv fragments with and without melittin signal peptide and detected that translocated scFv fragments were highly functional, whereas scFvs synthesized in the cytosol of the cell extract showed strongly decreased binding capabilities. Additionally, we describe a cell-free protein synthesis method for the incorporation of noncanonical amino acids into scFv molecules in eukaryotic cell lysates. We demonstrate the successful cotranslational labeling of de novo synthesized scFv molecules with fluorescent amino acids, using residue-specific as well as site-specific labeling.

Synthetic biology is a new and evolving branch of science, which has extensive application in the surrounding as well as human life. This field deals with the knowledge gain from the living system by the means of bioinformatics or other relevant field and try to regulate or restructure the pathways and the system of the higher organism in much simpler microorganism. It actually translates the knowledge gain from an organism or system in simpler system for the benefit of the nature. Evolution in the field of biotechnology, easy fast and high-throughput and accurate technologies available for DNA sequencing, and synthesis, has made it very easy to design and structure a specific pathway, which is useful in a particular organism in a model organism, which in field of synthetic biology is termed as chassis. Synthetic biology brings together different areas such as engineering, molecular biology, cell biology, biotechnology, bioinformatics, and system biology in such a way that they all together forms this new area with vast application in various fields. Synthetic biotechnology has its application in almost all fields such as from cellular programming to drug designing to biofuels production. In this chapter, we will be mainly dealing with the application part of the synthetic biotechnology in development of biosensors, for new drug discovery, tools for application in agriculture, for secondary metabolism, for chemical production, and for biofuel production.

Chapter 24 - Cell-free synthetic biology as an emerging biotechnology

Cell membranes are essential part of living cells. They are important as the envelope which encapsulate the biochemical systems within them and distinguish “self” components from “not-self” surrounding environment. Furthermore, cell membranes function as an interface which exchange materials between inside and outside of the cell, sense external environment, and transmit signals to inside systems to response the circumstance. In the field of synthetic biology, liposome (lipid membrane vesicle) has been widely used as a model of cell membrane. Although liposome is certainly a good model as for cell envelope, it is not satisfying the biochemical functions of cell membranes. Since the most of cell membrane functions are led by membrane embeded proteins, we should combine membrane proteins with liposome to construct more feasible artificial cell membranes. In this research, we aim to equip membrane machinery on liposome membrane using in vitro gene expression system. We show that a membrane machinery Sec translocon 1 , which conducts membrane secretion and insertion of protein (Figure 1), has been synthesized onto the liposome membrane from its template DNAs. The gene expression was performed with the cell-free protein synthesis system, PURE system 2 . The PURE system is a reconstructed transcription/translation system that actualizes the phenomenon of Central Dogma (DNA-RNA-protein) in vitro with the minimal number of factors. Synthesized Sec proteins spontaneously localized at lipid bilayer and about 80nM Sec translocon were produced in functional state. This indicates that 2-3 Sec translocon were allocated to one liposome membrane based on a sequence of statistical calculations. Although the population density of the produced Sec translocon was not so high, a substantial peptides secretion activity of the Sec translocon was detected by biochemical assays. The specific activity of the synthesized Sec translocon was comparable to that of native Sec translocon isolated from cells in the function of protein secretion. In addition, the synthesized Sec translocon was able to conduct membrane insertion of a multi-spanning protein. These results indicate that the artificially synthesized Sec translocon is functional both in secretion and insertion. It should be noted that the formation of Sec translocon was achieved in self-assembly process. Our results demonstrate that the functional Sec translocon has been constructed in totally synthetic manner. Although the protein synthesis in this study were performed on the outside of the liposomes, the same reaction would be occurred inside liposomes, for instance giant unilamellar vesicles that can effectively encapsulate a cell-free system and DNA 3 . More importantly, our results raise a possibility that various membrane proteins can be subsequently produced in liposome membrane by primarily constructed Sec translocon, and eventually non-functional liposomes will gain divers biofunctions that are essential for a living artificial cell.

Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development.

Chapter 20 - Research Trends in Genetically Modified (GM) Plants

Metabolic pathway engineering: Perspectives and applications

Cell-free protein synthesis (CFPS) is an effective method for the site-specific incorporations of noncanonical amino acids (ncAAs) into proteins. The nature of in vitro synthesis enables the use of experimental conditions that are toxic or reduce cellular uptake during in vivo site-specific incorporations of ncAAs. Using the Escherichia coli cell extract (S30) from the highly reproductive RF-1 deletion strains, B-60.∆A::Z and B-95.∆A, with orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) pairs from Methanosarcina mazei, we have developed CFPS methods for the highly productive and efficient multiple incorporation of ncAAs. In this chapter, we describe our methods for the preparation of the S30 and the orthogonal tRNAPyl and PylRS pair, and two CFPS protocols for ncAA incorporation.

Synthetic biology is an emerging and rapidly expanding field of research focused on the assembly of novel biological systems with new functionalities tailored for different applications. Genetic circuits have been re-wired or constructed with elements from different organisms, and metabolic pathways have been engineered to endow cells with non-natural capabilities. One of the most exciting goals of synthetic biology is bringing solutions to biomedical challenges. Though this research area is still in its infancy, translational medicine is already witnessing the first steps towards the development of therapies based on synthetic biology. Cell-free synthetic biology (or in vitro synthetic biology), a branch of synthetic biology, makes use of cell-free gene expression systems to create biological networks that operate outside the chassis of a living cell. More than a decade ago, the convergence of synthetic biology, cell-free gene expression systems and liposome technology gave rise to the creation of artificial vesicle bioreactors that can synthesize genetically-encoded molecules. Though the primary motivation of these studies was the assembly of a semi-synthetic cell, the application of such technologies for different therapeutic purposes has been envisioned. Examples of this are the creation of antigen-producing liposomes as novel vaccination systems, the development of bioreactor liposomes suited for remotely controlled in-situ mRNA or protein production, and the assembly of a PCR-based nanofactory for gene delivery. Liposomes are the most successful drug delivery scaffolds ever developed with more than fifteen liposome-based drugs approved. Liposomes have long been investigated in the field of gene therapy as delivery vehicles for nucleic acids to overcome the barriers encountered by these molecules in vivo. In particular, the field of RNA interference-based drugs is very promising, with the first marketed formulation in 2018, and several others on their way. However, despite their long history of investigation, most of the structural and functional properties of the liposome-based drug delivery systems are inferred from bulk measurements. Therefore, the level of heterogeneity regarding, e.g. encapsulation efficiency, lipid composition, remains largely unknown within liposome preparations, which complicates the development of new formulations with improved therapeutic efficiency. Another important limitation is the observed membrane leakiness and premature drug release upon administration. Controlling the bio-distribution of a therapeutic drug is essential to minimize toxic side effects and enhance the efficiency of the treatment. To tackle this problem, the creation of targeting delivery systems with stimuli-responsive ability can improve the biodistribution profile of a drug and allow its delivery on demand. This work contributes to the convergence of the fields of synthetic biology, single-liposome biophysics and biomedicine, presenting different possible applications of vesicle bioreactors for the improvement of current RNA-based gene delivery systems.

E. coli‐based cell‐free protein synthesis (CFPS) is a flexible platform technology for on‐demand protein production that allows users to express traditionally intractable proteins, perform high‐throughput screening, and, with augmentation, supports applications such as metabolic engineering and genetic code expansion. The broad utility of the CFPS platform arises from the elimination of the cellular membrane and capture of transcription and translation machinery in vitro. This obviates the need for living cells and creates an open system for direct manipulation of the environment of protein production. However, broad dissemination to field and classroom applications remains limited, as CFPS is dependent on technical scientific expertise for proper reaction setup and laboratory infrastructure for proper storage of reagents. Here, we report our efforts to improve broad accessibility of CFPS by 1) simplifying reaction setup and 2) improving shelf stability of the cell‐extract at various storage conditions. In order to simplify reaction setup, which currently requires precise pipetting of 10 reagents, we have combined the CFPS reagents into two stable premixes. These premixes can be stored at −20 °C for at least 4 months and allow the user to pipette fewer times with larger volumes for more accurate reaction setup by non‐experts. To improve the shelf stability of cell extract, which contains the sensitive transcription and translation machinery and is traditionally stored at −80 °C, we have characterized the functional effects of 10 protein stabilizing additives such as sugars, osmolytes, surfactants, and molecular crowding agents. Tests of single additives and combinations of two or three have allowed us to identify the landscape of small molecule additives that can support a variety of storage conditions ranging from −80 °C to room temperature to suit a variety of applications. When combined, these advances to reaction setup and cell extract composition provide the foundation for broad implementation of CFPS. Improved ease of reaction setup enables the use of CFPS by non‐experts, such as early career students, and improves efficiency for researchers and field applications. Additionally, increased storage stability diminishes reliance on the cold chain, making CFPS reagents easier to disseminate, commercialize, and use in the field. Together, these modifications provide a foundation for democratization of the CFPS platform.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology, Bill and Linda Frost Fund, and NSF‐1708919.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.