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Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3\u00a0\u00d7\u00a010<jats:sup>\u20133<\/jats:sup> and 13.2\u00a0\u00d7\u00a010<jats:sup>\u20133<\/jats:sup>, depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in <jats:italic>E. coli<\/jats:italic>. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system.<\/jats:p>","DOI":"10.1038\/s41598-020-80827-8","type":"journal-article","created":{"date-parts":[[2021,1,21]],"date-time":"2021-01-21T11:03:39Z","timestamp":1611227019000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":49,"title":["In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity"],"prefix":"10.1038","volume":"11","author":[{"given":"Anne","family":"Doerr","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Foschepoth","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony C.","family":"Forster","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christophe","family":"Danelon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,1,21]]},"reference":[{"key":"80827_CR1","doi-asserted-by":"publisher","first-page":"387","DOI":"10.1038\/35053176","volume":"409","author":"JW Szostak","year":"2001","unstructured":"Szostak, J. 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