PXD017584 is an
original dataset announced via ProteomeXchange.
Dataset Summary
Title | Protocol for high-yield production of photo-leucine-labeled proteins in Escherichia coli |
Description | Cross-linking mass spectrometry (XL-MS) is an emerging technique to obtain structural information of biomacromolecules and their complexes in cells and in vitro. In particular, certain photo-reactive amino acids (pA) such as photo-leucine (pLeu) and photo-methionine can provide unique short-distance information on structural core regions of proteins. Here, we present a protocol for high-yield incorporation of pLeu in proteins recombinantly expressed in Escherichia coli. The protein is expressed at high cell densities, which reduces the required amount of the photo-reactive amino acid by a factor of 10 compared to standard protocols, while maintaining high incorporation rates. For the two chaperones Trigger Factor and SecB, up to 3 mg of photo-leucine labeled protein were thus obtained from 100 ml of cell culture, with label incorporation rates of up to 34%. For Trigger factor, UV-induced cross-linking lead to the identification of 12 cross-links that are in agreement with published three-dimensional structures. The accessibility of milligram amounts of photo-leucine-labeled proteins at low costs will be highly useful to address structural biology questions. |
HostingRepository | PRIDE |
AnnounceDate | 2020-05-26 |
AnnouncementXML | Submission_2020-05-26_05:24:45.xml |
DigitalObjectIdentifier | |
ReviewLevel | Peer-reviewed dataset |
DatasetOrigin | Original dataset |
RepositorySupport | Unsupported dataset by repository |
PrimarySubmitter | Bastian Kohl |
SpeciesList | scientific name: Escherichia coli; NCBI TaxID: 562; |
ModificationList | carbamoylated residue |
Instrument | Orbitrap Fusion Lumos; LTQ Orbitrap Elite |
Dataset History
Revision | Datetime | Status | ChangeLog Entry |
0 | 2020-02-18 05:21:57 | ID requested | |
⏵ 1 | 2020-05-26 05:24:46 | announced | |
Publication List
Kohl B, Br, ΓΌ, derlin M, Ritz D, Schmidt A, Hiller S, . J Proteome Res, 19(8):3100-3108(2020) [pubmed] |
Keyword List
submitter keyword: Photo-leucine, Mass spectrometry, high-cell density culture, UV cross-linking, Molecular chaperones |
Contact List
Sebastian Hiller |
contact affiliation | University of Basel Biozentrum FG Sebastian Hiller Klingelbergstrasse 70 4056 Basel |
contact email | sebastian.hiller@unibas.ch |
lab head | |
Bastian Kohl |
contact affiliation | University of Basel Biozentrum FG Sebastian Hiller Klingelbergstrasse 70 4056 Basel |
contact email | bastian.kohl@unibas.ch |
dataset submitter | |
Full Dataset Link List
Dataset FTP location
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PRIDE project URI |
Repository Record List
[ + ]
[ - ]
- PRIDE
- PXD017584
- Label: PRIDE project
- Name: Protocol for high-yield production of photo-leucine-labeled proteins in Escherichia coli