The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/ 05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.

Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells. It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system. This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels, including protein engineering, metabolic engineering, and artificial cell engineering. In cell-free protein engineering, the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins, toxic proteins, membrane proteins, and novel proteins with unnatural amino acids. Cell-free systems offer the ability to design metabolic pathways towards the production of desired products. Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.

Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.virus-like particle | engineered nanoparticles | disulfide stabilization | hepatitis core protein | cell-free protein synthesis V irus-like particles (VLPs) are probably the most precisely defined and, therefore, potentially the most useful complex nanometer-scale scaffolds (1). VLPs mimic the capsid structure of real viruses, but lack infectious genetic material. Selected VLPs derived from pathogens have already provided major advances in the development of vaccines that have known and relatively homogeneous structures as well as enhanced immunogenicity (2). Such nanoparticles provide comparable cellular uptake and intracellular trafficking compared with natural viruses (3), and also have repetitive surfaces for the high-density display of vaccine antigens (4). In addition, VLPs offer favorable trafficking from the injection site to lymph nodes (5). Since the first reported use of a hepatitis B core protein (HBc) VLP as an antigen carrier in 1987 (6), at least 110 VLP vaccine candidates have been constructed by using capsid proteins from 35 different viral families (7).Among different types of VLPs, the HBc VLP is the most flexible and promising model for fundamental and applied immunological studies (8). One advantage of the HBc VLP platform is that the capsids can be produ...

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Background: In addition to regulating blood pressure and body fluid homeostasis, the renin-angiotensin system is also involved in lung fibrogenesis. Objective: To study the effect of losartan, an angiotensin II antagonist, on bleomycin-induced pulmonary fibrosis in rats and its possible mechanism. Methods: Pulmonary fibrosis was induced in SD rats by intratracheal instillation of bleomycin (5 mg·kg^–1). Subsequently, the rats received daily losartan (3, 9 and 27 mg·kg^–1) or prednisone (20 mg·kg^–1) orally. Six rats in each group were sacrificed 14 and 21 days after intratracheal instillation. Hydroxyproline, superoxide dismutase (SOD), and malondialdehyde (MDA) levels in lung tissues were determined by spectroscopy. The levels of TGF-β1 in serum were measured by ELISA. Histological changes in the lungs were evaluated by hematoxylin-eosin stain, and scored. Results: Rat body weight evidently decreased while the indices of lung and hydroxyproline contents in lung tissue were significantly increased 14 and 21 days after intratracheal bleomycin instillation. Inflammatory cell infiltration and fibrotic scores were more prominent in the model group compared to the sham group. Losartan (3, 9 and 27 mg·kg^–1, i.g.) apparently attenuated the degree of pulmonary fibrosis. Further study showed that losartan significantly increased SOD levels while it decreased MDA contents in lung homogenates. Serum TGF-β1 levels of pulmonary fibrosis rats were also decreased by losartan. Conclusions: Losartan had an inhibitory effect on bleomycin-induced pulmonary fibrosis, and its effect may be associated with its anti-free radicals and the reduction in TGF-β1.

Building an artificial cell is a research area that is rigorously studied in the field of synthetic biology. It has brought about much attention with the aim of ultimately constructing a natural cell-like structure. In particular, with the more mature cell-free platforms and various compartmentalization methods becoming available, achieving this aim seems not far away. In this review, we discuss the various types of artificial cells capable of hosting several cellular functions. Different compartmental boundaries and the mature and evolving technologies that are used for compartmentalization are examined, and exciting recent advances that overcome or have the potential to address current challenges are discussed. Ultimately, we show how compartmentalization and cell-free systems have, and will, come together to fulfill the goal to assemble a fully synthetic cell that displays functionality and complexity as advanced as that in nature. The development of such artificial cell systems will offer insight into the fundamental study of evolutionary biology and the sea of applications as a result. Although several challenges remain, emerging technologies such as artificial intelligence also appear to help pave the way to address them and achieve the ultimate goal.

Incorporation of unnatural amino acids (UNAAs) into proteins currently is an active biological research area for various fundamental and applied science. In this context, cell-free synthetic biology (CFSB) has been developed and recognized as a robust testing and biomanufacturing platform for highly efficient UNAA incorporation. It enables the orchestration of unnatural biological machinery toward an exclusive user-defined objective of unnatural protein synthesis. This review aims to overview the principles of cell-free unnatural protein synthesis (CFUPS) systems, their advantages, different UNAA incorporation approaches, and recent achievements. These have catalyzed cutting-edge research and diverse emerging applications. Especially, present challenges and future trends are focused and discussed. With the development of CFSB and the fusion with other advanced next-generation technologies, CFUPS systems would explicitly deliver their values for biopharmaceutical applications.