title: N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions.
abstract: 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.
authors.affiliation: Department of Computational and Structural Biology, F. Max Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria.
authors.forename: Andrea Gabriele
authors.initials: AG
authors.lastname: Flamm
authors.affiliation: 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.
authors.forename: Anabel-Lise
authors.initials: AL
authors.lastname: Le Roux
authors.affiliation: Biomolecular NMR Laboratory, Department of Organic Chemistry, University of Barcelona, Baldiri Reixac 10-12, 08028, Barcelona, Spain.
authors.forename: Borja
authors.initials: B
authors.lastname: Mateos
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10-12, 08028, Barcelona, Spain.
authors.forename: Mireia
authors.initials: M
authors.lastname: Díaz-Lobo
authors.affiliation: Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, CCB, Innrain 80/82, 6020, Innsbruck, Austria.
authors.forename: Barbara
authors.initials: B
authors.lastname: Storch
authors.affiliation: Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, CCB, Innrain 80/82, 6020, Innsbruck, Austria.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Breuker
authors.affiliation: Department of Computational and Structural Biology, F. Max Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria.
authors.forename: Robert
authors.initials: R
authors.lastname: Konrat
authors.affiliation: Biomolecular NMR Laboratory, Department of Organic Chemistry, University of Barcelona, Baldiri Reixac 10-12, 08028, Barcelona, Spain.
authors.forename: Miquel
authors.initials: M
authors.lastname: Pons
authors.affiliation: 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.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Coudevylle
mesh_terms: D019814:Myristic Acid / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D012996:Solutions; D019814:Myristic Acid
keywords: intrinsically disordered proteins; lauroylation; myristoylation; surface plasmon resonance; transferases