Protocol for High-Yield Production of Photo-Leucine-Labeled Proteins in Escherichia coli.
J Proteome Res
; 19(8): 3100-3108, 2020 08 07.
Article
em En
| MEDLINE
| ID: mdl-32412763
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
Escherichia coli
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article