PXD020877-2
PXD020877 is an original dataset announced via ProteomeXchange.
Dataset Summary
Title | In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity |
Description | The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. 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 × 10–3 and 13.2 × 10–3, 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 E. coli. 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. |
HostingRepository | PanoramaPublic |
AnnounceDate | 2021-09-06 |
AnnouncementXML | Submission_2021-09-06_17:53:19.949.xml |
DigitalObjectIdentifier | |
ReviewLevel | Peer-reviewed dataset |
DatasetOrigin | Original dataset |
RepositorySupport | Supported dataset by repository |
PrimarySubmitter | David Foschepoth |
SpeciesList | scientific name: Escherichia coli; NCBI TaxID: 562; |
ModificationList | Label:15N(1); Label:15N(2); Label:15N(4); Label:15N(3) |
Instrument | 6460 Triple Quadrupole LC/MS |
Dataset History
Revision | Datetime | Status | ChangeLog Entry |
---|---|---|---|
0 | 2020-08-12 21:38:18 | ID requested | |
1 | 2021-09-06 17:45:32 | announced | |
⏵ 2 | 2021-09-06 17:53:20 | announced | : updated lab head |
Publication List
Doerr A, Foschepoth D, Forster AC, Danelon C, In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity. Sci Rep, 11(1):1898(2021) [pubmed] |
Keyword List
submitter keyword: PURE, synthetic cell, ribosome processivity, gene expression, in vitro gene expression, cell free, in vitro evolution |
Contact List
Christophe Danelon | |
---|---|
contact affiliation | TU Delft |
contact email | c.j.a.danelon@tudelft.nl |
lab head | |
David Foschepoth | |
contact affiliation | TU Delft |
contact email | d.j.foschepoth@tudelft.nl |
dataset submitter |
Full Dataset Link List
Panorama Public dataset URI |