PXD024458

PXD024458 is an original dataset announced via ProteomeXchange.

Title	Preparation of a high expression yield bacterial cell extract via lysozyme-assisted sonication
Description	One of the central goals for the development of cell-free (CF) gene expression systems is to ensure reproducible expression of proteins at high quality and yield. Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols have been developed and optimized reaction conditions have been established. One of the crucial steps during the preparation of cell extract-based expression systems, however, is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here, we demonstrate for an E. coli based system that a lysis procedure combining incubation of the cells with lysozyme with a gentle sonication step results in highly active cell extracts. As examples for the capabilities of our extract, we demonstrate the production of several fluorescent proteins as well as the production and assembly of T7 bacteriophages. Compared to other cell-free expression systems, our method achieved the highest phage titers in our plaque assays. Stateof-the-art quantitative proteomics revealed that enzymes of the energy regeneration pathway were enriched in our extract compared to a widely used commercial cell extract. On the other hand, ribosomal proteins were found to be more abundant in the commercial product.
HostingRepository	PRIDE
AnnounceDate	2021-08-05
AnnouncementXML	Submission_2021-08-05_02:34:50.250.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Christina Ludwig
SpeciesList	scientific name: Escherichia coli BL21(DE3); NCBI TaxID: 469008;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2021-03-02 06:32:08	ID requested
⏵ 1	2021-08-05 02:34:50	announced

Falgenhauer E, von Sch, ö, nberg S, Meng C, M, ü, ckl A, Vogele K, Emslander Q, Ludwig C, Simmel FC, Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics. Chembiochem, 22(18):2805-2813(2021) [pubmed]

submitter keyword: Cell-free protein expression, bacterial cell extract, in vitro transcription-translation, synthetic biology, phage assembly

Christina Ludwig
contact affiliation	Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Freising, TU Munich
contact email	tina.ludwig@tum.de
lab head
Christina Ludwig
contact affiliation	TU Munich
contact email	tina.ludwig@tum.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD024458
     1. Label: PRIDE project
     2. Name: Preparation of a high expression yield bacterial cell extract via lysozyme-assisted sonication