N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions.

Autor: Flamm AG; Department of Computational and Structural Biology, F. Max Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria., Le Roux AL; Biomolecular NMR Laboratory, Department of Organic Chemistry, University of Barcelona, Baldiri Reixac 10-12, 08028, Barcelona, Spain.; Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10-12, 08028, Barcelona, Spain., Mateos B; Biomolecular NMR Laboratory, Department of Organic Chemistry, University of Barcelona, Baldiri Reixac 10-12, 08028, Barcelona, Spain., Díaz-Lobo M; Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10-12, 08028, Barcelona, Spain., Storch B; Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, CCB, Innrain 80/82, 6020, Innsbruck, Austria., Breuker K; Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, CCB, Innrain 80/82, 6020, Innsbruck, Austria., Konrat R; Department of Computational and Structural Biology, F. Max Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria., Pons M; Biomolecular NMR Laboratory, Department of Organic Chemistry, University of Barcelona, Baldiri Reixac 10-12, 08028, Barcelona, Spain., Coudevylle N; Department of Computational and Structural Biology, F. Max Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria. nicolas.coudevylle@univie.ac.at.
Jazyk: angličtina
Zdroj: Chembiochem : a European journal of chemical biology [Chembiochem] 2016 Jan 01; Vol. 17 (1), pp. 82-9. Date of Electronic Publication: 2015 Dec 03.
DOI: 10.1002/cbic.201500454
Abstrakt: Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N-myristoylated proteins in Escherichia coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1-185 N-terminal region of c-Src, we show the significant, and protein-specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl-free samples of myristoylated proteins in both rich and minimal media.
(© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
Databáze: MEDLINE
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