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Protein Science (2007), 16:919-928. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Expression of proteins with dimethylarginines in Escherichia coli for protein–protein interaction studies

Cheng-Hsilin Hsieh1, San-Yuan Huang3,4, Yu-Ching Wu1, Li-Fan Liu1, Chau-Chung Han2, Yi-Chen Liu1, and Ming F. Tam1

1 Institute of Molecular Biology, Academia Sinica, Taiwan, Republic of China
2 Institute of Atomic and Molecular Sciences, Academia Sinica, Taiwan, Republic of China
3 Division of Biotechnology, Animal Technology Institute Taiwan, Taiwan, Republic of China
4 Department of Animal Science, National Chung Hsing University, Taiwan, Republic of China

(RECEIVED November 16, 2006; FINAL REVISION February 1, 2007; ACCEPTED February 22, 2007)

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}-NG,NG-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 His6-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.

Keywords: protein expression; protein arginine methylation; protein–protein interactions; Sbp1p; Stm1p


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