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Orders Norzivirales and Timlovirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
A highly specific approach for the further development of VLP vectors was achieved by the asymmetrization of the Qβ VLPs after the introduction of a single copy of the maturation protein A2, which allowed the production of VLPs with a single unique modification (Smith et al. 2012). This approach was driven by the estimations that, by bioconjugation described earlier, the theoretical maximum of the Qβ VLPs having a single molecule attached could be 37%, according to the Poisson probability distribution. The remaining VLP population would contain zero (37%) or multiple (26%) molecules attached to each VLP and would not be easily separated from the VLPs with one molecule. The E. coli-based cell-free protein synthesis system was employed for the coexpression of the cytotoxic A2 protein and the coat of the phage Qβ to form a nearly monodispersed population of novel VLPs. The cell-free protein synthesis allowed for direct access and optimization of both protein synthesis and VLP self-assembly. The A2 was shown to be incorporated at high efficiency, approaching a theoretical maximum of one A2 per VLP. This work demonstrated for the first time the de novo production of a novel VLP, which contained a unique site that could have the potential for future nanometric engineering applications (Smith et al. 2012).
Physiology and Growth
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Strong contribution of cell-free techniques to modern nanotechnology was achieved recently by Schwarz-Schilling et al. (2018). Gregorio et al. (2019) published an extensive overview of cell-free protein synthesis over 60 years.
The Use of Molecular Hybridization Techniques as Tools to Evaluate Hepatic Fibrogenesis
Published in Marcos Rojkind, Connective Tissue in Health and Disease, 2017
Mark Α. Zern, Mark J. Czaja, Francis R. Weiner
During the past 2 decades, progress in our understanding of the basic principles of nucleic acid structure and interaction has made it possible to use recombinant DNA probes to study gene organization and function. Molecular hybridization is a reaction between two molecules of DNA or one molecule of DNA and RNA, based on the ability of complementary nucleotides in these molecules to interact and form stable base pairs by hydrogen bonding (adenine [A] recognizes thymidine [T] or uracil [U], and guanine [G] recognizes cytosine [C]). This bonding is the same principle which forms the basis of the Watson-Crick structure of DNA, the "double helix". Molecular hybridization can precisely quantitate the content of a specific mRNA sequence and is not dependent on an assay requiring biological activity. This represents an advantage over assays utilizing cell-free protein synthesis, since the latter may be highly variable and is not quantitative.21,22
Full-length recombinant antibodies from Escherichia coli: production, characterization, effector function (Fc) engineering, and clinical evaluation
Published in mAbs, 2022
E. coli has unique features compared to most other production hosts. While recombinant proteins are usually secreted into the culture media in other hosts (e.g., mammalian or fungal systems), in E. coli, they are expressed in the cytoplasm, targeted to the periplasmic space, or secreted into the culture media41 (Figure 2). Each cellular compartment has unique properties and, based on the protein to be produced, a strategic decision can be made as to where to direct the recombinant protein. In the reducing cytoplasm, proteins can be produced in soluble form or as inclusion bodies (IBs), which can be resolubilized and refolded into functional forms. Proteins that require an oxidizing environment, for disulfide bond formation, can be secreted into the oxidizing periplasmic compartment in soluble and active forms. In some instances, proteins can also be secreted into the culture media for ease of downstream processing. In addition, cell-free protein synthesis (CFPS) systems using E. coli cell extracts or systems using purified components (the PURE system) have seen significant improvements, and are now competing with E. coli cell-based production systems.46,47
Advances of droplet-based microfluidics in drug discovery
Published in Expert Opinion on Drug Discovery, 2020
Yuetong Wang, Zhuoyue Chen, Feika Bian, Luoran Shang, Kaixuan Zhu, Yuanjin Zhao
Cell-free protein synthesis provides many advantages for high-throughput applications compared with cell culture–based protein synthesis, since it does not require the transformation or culturing of cells and thus greatly accelerates the screening of large protein libraries. Microfluidic technology can be used to further leverage these advantages and aid in high-throughput cell-free protein synthesis. In vitro protein synthesis in a droplet-based microfluidic system was recently demonstrated by Mazutis et al. [96] by combining both on-chip and off-chip operations. This system was used for in vitro transcription and translation of Bacillus subtilis cotA laccase genes and kinetic analysis of the catalytic activity of the translated protein.
Malaria transmission-blocking vaccines: wheat germ cell-free technology can accelerate vaccine development
Published in Expert Review of Vaccines, 2019
Kazutoyo Miura, Mayumi Tachibana, Eizo Takashima, Masayuki Morita, Bernard N. Kanoi, Hikaru Nagaoka, Minami Baba, Motomi Torii, Tomoko Ishino, Takafumi Tsuboi
Effective protein expression systems with their proper folding are essential for production and use of proteins in biochemical and biomedical research including malaria vaccine research. Among the different approaches used for protein synthesis, cell-free systems have gained high attention for their ability to rapidly produce proteins under controlled conditions in the post-genome era [80]. While driving the progress of cell-free protein expression, E. coli-derived expression systems have failed, in many cases, to express the proteins in full-length, or in soluble forms. Moreover, E. coli-derived systems may not properly fold proteins of the eukaryotic targets [80]. Hence, E. coli-expression systems often yield inactive, misfolded or truncated proteins including those from malaria parasites [81]. In contrast, the wheat germ cell-free protein synthesis system (WGCFS) has successfully expressed a wide range of eukaryotic proteins, including complex proteins, with good yield, indicating that WGCFS is the method of choice for production of stable, properly folded proteins and for high-throughput protein expression at various scales. The advantages of WGCFS over other protein expression systems had been discussed intensively [80,82–84]. The high performance of WGCFS has been further demonstrated in the so-called ‘human protein factory’ study [85]. The project targeted the expression of 13,364 human proteins, where 12,996 of their clones produced protein in the WGCFS (97.2%); of those, 12,682 were found in the soluble fraction. Hence, WGCFS can be a very effective high-throughput expression system for rapid preparation of malaria parasite proteins for antigen discovery, characterization, and generation of quality antibodies [86,87].