A new protein engineering approach combining chemistry and biology, part I; site-specific incorporation of 4-iodo-L-phenylalanine in vitro by using misacylated suppressor tRNAPhe.

Koichiro Kodama, Seketsu Fukuzawa, Kensaku Sakamoto, Hiroshi Nakayama, Takanori Kigawa, Takashi Yabuki, Natsuko Matsuda, Mikako Shirouzu, Koji Takio, Kazuo Tachibana, Shigeyuki Yokoyama

Department of Biophysics and Biochemistry, School of Science The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.

Chembiochem : a European journal of chemical biology 2006 Oct

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An Escherichia coli suppressor tRNA(Phe) (tRNA(Phe) (CUA)) was misacylated with 4-iodo-L-phenylalanine by using the A294G phenylalanyl-tRNA synthetase mutant (G294-PheRS) from E. coli at a high magnesium-ion concentration. The preacylated tRNA was added to an E. coli cell-free system and a Ras protein that contained the 4-iodo-L-phenylalanine residue at a specific target position was synthesized. Site-specific incorporation of 4-iodo-L-phenylalanine was confirmed by using LC-MS/MS. Free tRNA(Phe) (CUA) was not aminoacylated by aminoacyl-tRNA synthetases (aaRSs) present in the E. coli cell-free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins.

Citation

Koichiro Kodama, Seketsu Fukuzawa, Kensaku Sakamoto, Hiroshi Nakayama, Takanori Kigawa, Takashi Yabuki, Natsuko Matsuda, Mikako Shirouzu, Koji Takio, Kazuo Tachibana, Shigeyuki Yokoyama. A new protein engineering approach combining chemistry and biology, part I; site-specific incorporation of 4-iodo-L-phenylalanine in vitro by using misacylated suppressor tRNAPhe. Chembiochem : a European journal of chemical biology. 2006 Oct;7(10):1577-81