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  Selective methyl labeling of eukaryotic membrane proteins using cell-free expression.

Linser, R., Gelev, V., Hagn, F., Arthanari, H., Hyberts, S. G., & Wagner, G. (2014). Selective methyl labeling of eukaryotic membrane proteins using cell-free expression. Journal of the American Chemical Society, 136(32), 11308-11310. doi:10.1021/ja504791j.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0019-D54D-6 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-3AF5-A
Genre: Journal Article

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http://pubs.acs.org/doi/pdf/10.1021/ja504791j (Publisher version)
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Linser, R.1, Author              
Gelev, V., Author
Hagn, F., Author
Arthanari, H., Author
Hyberts, S. G., Author
Wagner, G., Author
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1Research Group of Solid-State NMR-2, MPI for Biophysical Chemistry, Max Planck Society, ou_1950286              

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 Abstract: Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins.

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Language(s): eng - English
 Dates: 2014-06-172014
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1021/ja504791j
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Title: Journal of the American Chemical Society
Source Genre: Journal
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Pages: - Volume / Issue: 136 (32) Sequence Number: - Start / End Page: 11308 - 11310 Identifier: -