PXD020877

PXD020877 is an original dataset announced via ProteomeXchange.

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

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

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]

submitter keyword: PURE, synthetic cell, ribosome processivity, gene expression, in vitro gene expression, cell free, in vitro evolution

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

Panorama Public dataset URI