Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system.
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.
Suzuki
Takashi
T
Clinical and Biotechnology Business Unit, Shimadzu Corporation, Kyoto, Japan.
Moriya
Koko
K
Nagatoshi
Kei
K
Ota
Yoshinobu
Y
Ezure
Toru
T
Ando
Eiji
E
Tsunasawa
Susumu
S
Utsumi
Toshihiko
T
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System / Q000737:chemistry; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019814:Myristic Acid / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D018076:DNA, Complementary; D011506:Proteins; D019814:Myristic Acid