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

Abstract

An Escherichia coli suppressor tRNAPhe (tRNAPheCUA) was misacylated with 4-iodo-L-phenytalanine by using the A294C phenyl-alanyl-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 tRNAPheCUA 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. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.