title: Expression of proteins with dimethylarginines in Escherichia coli for protein-protein interaction studies.
abstract: Protein arginine methylation often modulates protein-protein interactions. To isolate a sufficient quantity of proteins enriched in methyl arginine(s) from natural sources for biochemical studies is laborious and difficult. We describe here an expression system that produces recombinant proteins that are enriched in omega-N(G),N(G)-asymmetry dimethylarginines. A yeast type I arginine methyltransferase gene (HMT1) is put on a plasmid under the control of the Escherichia coli methionine aminopeptidase promoter for constitutive expression. The protein targeted for post-translational modification is put on the same plasmid behind a T7 promoter for inducible expression of His(6)-tagged proteins. Sbp1p and Stm1p were used as model proteins to examine this expression system. The 13 arginines within the arginine-glycine-rich motif of Sbp1p and the RGG sequence near the C terminus of Stm1p were methylated. Unexpectedly, the arginine residue on the thrombin cleavage site (LVPRGS) of the fusion proteins can also be methylated by Hmt1p. Sbp1p and Sbp1p/hmt1 were covalently attached to solid supports for the isolation of interacting proteins. The results indicate that arginine methylation on Sbp1p exerts both positive and negative effects on protein-protein interaction.
authors: Institute of Molecular Biology, Academia Sinica, Taiwan, Republic of China.
authors: Cheng-Hsilin
authors: CH
authors: Hsieh
authors: San-Yuan
authors: SY
authors: Huang
authors: Yu-Ching
authors: YC
authors: Wu
authors: Li-Fan
authors: LF
authors: Liu
authors: Chau-Chung
authors: CC
authors: Han
authors: Yi-Chen
authors: YC
authors: Liu
authors: Ming F
authors: MF
authors: Tam
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008745:Methylation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005656:Fungal Proteins; D011506:Proteins; D011994:Recombinant Proteins; C487735:dimethylarginine; D001120:Arginine; D011484:Protein-Arginine N-Methyltransferases