Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system.
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Suzuki T, Moriya K, Nagatoshi K, Ota Y, Ezure T, Ando E, Tsunasawa S, Utsumi T
Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system.
Proteomics. 2010 May;10(9):1780-93. doi: 10.1002/pmic.200900783.
- PubMed ID
- 20213681 [ View in PubMed]
- Abstract
To establish a strategy for the comprehensive identification of human N-myristoylated proteins, the susceptibility of human cDNA clones to protein N-myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell-free protein synthesis system. One-hundred-and-forty-one cDNA clones with N-terminal Met-Gly motifs were selected as potential candidates from approximately 2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N-myristoylation was evaluated using fusion proteins, in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N-myristoylated. The metabolic labeling experiments both in an insect cell-free protein synthesis system and in the transfected COS-1 cells using full-length cDNA revealed that 27 out of 29 proteins were in fact N-myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N-myristoylated proteins that have not been reported previously to be N-myristoylated, indicating that this strategy is useful for the comprehensive identification of human N-myristoylated proteins from human cDNA resources.