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Nucleic Acids Res. 2019 Feb 6. doi: 10.1093/nar/gkz069. [Epub ahead of print]

C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system.

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Eukarÿs SAS, Génopole Campus 3, 4 rue Pierre Fontaine, 91058 Evry Cedex, France.
Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257 CNRS/AMU, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France.
Institut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-Saclay, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Sorbonne Université, CNRS-UMR8256, Biological Adaptation and Ageing, Institut de Biologie Paris Seine (B2A-IBPS), F-75252 Paris, France.
Medical Microbiology and Immunology, University of Alberta, 6-142J Katz Group Centre for Pharmacy and Health Research, 114 Street NW, Edmonton, Alberta T6G 2E1, Canada.
INSERM Unit 1016, Institut Cochin, Bâtiment Gustave Roussy, 27 rue du faubourg Saint-Jacques, 75014 Paris, France.
Gastrointestinal and Metabolic Dysfunctions in Nutritional Pathologies, INSERM UMRS1149, 16 rue Henri Huchard, 75890 Paris Cedex 18, France.


Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.


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