Detection of co- and posttranslational protein N-myristoylation by metabolic labeling in an insect cell-free protein synthesis system.

To establish a simple and sensitive method to detect protein N-myristoylation, the usefulness of a newly developed cell-free protein synthesis system derived from insect cells for detecting protein N-myristoylation by in vitro metabolic labeling was examined. The results showed that in vitro translation of cDNA coding for N-myristoylated protein in the presence of [(3)H]myristic acid followed by SDS-PAGE and fluorography is a useful method for rapid detection of protein N-myristoylation. Differential labeling of N-myristoylated model proteins with [(3)H]leucine, [(3)H]myristic acid, and [(35)S]methionine revealed that the removal of the initiating Met during the N-myristoylation reaction could be detected using this system. Analysis of the N-myristoylation of a series of model proteins with mutated N-myristoylation motifs revealed that the amino acid sequence requirements for the N-myristoylation reaction in this system are quite similar to those observed in the rabbit reticulocyte lysate system. N-myristoylation of tBid (a posttranslationally N-myristoylated cytotoxic protein that could not be expressed in transfected cells) was successfully detected in this assay system. Thus, metabolic labeling in an insect cell-free protein synthesis system is an effective strategy to detect co- and posttranslational protein N-myristoylation irrespective of the cytotoxicity of the protein.

Citation

Nagisa Sakurai, Koko Moriya, Takashi Suzuki, Kozue Sofuku, Hiroyuki Mochiki, Osamu Nishimura, Toshihiko Utsumi. Detection of co- and posttranslational protein N-myristoylation by metabolic labeling in an insect cell-free protein synthesis system. Analytical biochemistry. 2007 Mar 15;362(2):236-44

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PMID: 17266917

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