title: Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry.
abstract: A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.
authors.affiliation: Department of Chemistry, Imperial College London, UK.
authors.forename: William P
authors.initials: WP
authors.lastname: Heal
authors.forename: Megan H
authors.initials: MH
authors.lastname: Wright
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Thinon
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate
mesh_terms: D000217:Acyltransferases / Q000378:metabolism; D057930:Click Chemistry; D004926:Escherichia coli / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D011506:Proteins; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase