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N‐Lauroylation during the Expression of Recombinant N‐Myristoylated Proteins: Implications and Solutions
Author(s) -
Flamm Andrea Gabriele,
Le Roux AnabelLise,
Mateos Borja,
DíazLobo Mireia,
Storch Barbara,
Breuker Kathrin,
Konrat Robert,
Pons Miquel,
Coudevylle Nicolas
Publication year - 2016
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500454
Subject(s) - myristic acid , myristoylation , recombinant dna , biochemistry , chemistry , escherichia coli , lauric acid , fusion protein , fatty acid , membrane , palmitic acid , gene
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.