Protocol for High-Yield Production of Photo-Leucine-Labeled Proteins in Escherichia coli

Kohl, Bastian and Brüderlin, Mitchell and Ritz, Danilo and Schmidt, Alexander and Hiller, Sebastian. (2020) Protocol for High-Yield Production of Photo-Leucine-Labeled Proteins in Escherichia coli. Journal of Proteome Research, 19 (8). pp. 3100-3108.

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Official URL: https://edoc.unibas.ch/79813/

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UV-cross-linking mass spectrometry is an emerging technique to obtain structural information of biomacromolecules and their complexes; in vivo; 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 the 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 of interest is expressed at high cell densities, which reduces the required amount of the pA by a factor of 10, as compared to the standard protocols, while maintaining high incorporation rates. For the two chaperones, trigger factor and SecB, up to 3 mg of pLeu-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 leads to the identification of 12 cross-links that are in agreement with the published three-dimensional structures. The accessibility of milligram amounts of pLeu-labeled proteins at low costs will be highly useful to address structural biology questions.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Services Biozentrum > Proteomics (Schmidt)
05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Hiller)
UniBasel Contributors:Hiller Odermatt, Sebastian and Schmidt, Alexander
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:American Chemical Society
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:08 Dec 2022 10:21
Deposited On:31 Jan 2022 08:34

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