Strategy for comprehensive identification of human N‐myristoylated proteins using an insect cell‐free protein synthesis system

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.

Protein N-Myristoylation Is Required for Cellular Morphological Changes Induced by Two Formin Family Proteins, FMNL2 and FMNL3

“…0 -GCATGCGATCGCCAT-GGGCAACCTGGAGAGCGCC-3 0 and FXC1701-PmeI C, 5 0 -ATAT-GTTTAAACCGTGGGGGCCTGGAGT-3 0 , as primers, with pcDNA3-FXC1701-FLAG 11) as template. Plasmid pcDNA3-EGFP, containing the cDNA coding for EGFP, was constructed by PCR using two oligonucleotides, EcoRV-EGFP N, 5…”

“…0 -ATATGCGA-TCGCGATGGCAAACGCAGGGAGCATG-3 0 and FM2-Ástop-PmeI C, 5 0 -GCGCGTTTAAACCATTGTTATTTCGGC-3 0 , as primers, pcDNA3-FXC1184 11) as template. After digestion with SgfI and PmeI, the amplified fragments were subcloned into the SgfI-EcoRV sites of pcDNA3-FL.…”

“…Plasmid pcDNA3-FL, used as the mammalian expression vector, was constructed as described previously. 11) To construct pcDNA3 plasmids containing full-length KOP cDNA clones, pcDNA3-FL was treated with SgfI and EcoRV, and KOP cDNA clones digested with SgfI and PmeI were subcloned into the vector. Plasmid pcDNA3-FXC00892-G2A-Flag was constructed by polymerase chain reaction (PCR) using two oligonucleotides, SgfI-FXC00892-G2A N, 5…”

Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues

“…The results showed that in vitro translation of complementary DNA (cDNA) coding for N-myristoylated protein in the presence of [ 3 H] myristic acid followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography is a useful method for the detection of protein N-myristoylation [18][19][20]. However, this strategy, like other approaches, is still in need of improvement.…”

The Protein Lipidation and Its Analysis

“…Similarly, the heterogenicity of the lipid groups present in the GAP-43 protein, which has two acylated cysteines near the C-terminus, was studied by MALDI-TOF and confirmed by Quadropole-TOF MS/MS indicating that not only palmitoyl but also stearic acid can be attached on these cysteines [51]. Recently, MALDI-MS was also used for the detection of purified myristoylated and isoprenylated proteins generated in an insect-cell free protein synthesis system [88][89][90]. However, one of the limitations of this approach is the high hydrophobicity of the lipidated samples, which usually results in low efficiency of peptide recovery during sample preparation as well as peptide detection.…”