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
| Autor: | Hiroshi Nakayama, Koichiro Kodama, Takashi Yabuki, Natsuko Matsuda, Kensaku Sakamoto, Mikako Shirouzu, Takanori Kigawa, Seketsu Fukuzawa, Shigeyuki Yokoyama, Koji Takio, Kazuo Tachibana |
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| Rok vydání: | 2006 |
| Předmět: |
Cell-Free System
Chemistry Acylation Phenylalanine Organic Chemistry Mutant RNA Protein engineering Protein Engineering medicine.disease_cause Biochemistry Cell-free system RNA Transfer Phe Residue (chemistry) RNA Transfer Transfer RNA Escherichia coli ras Proteins medicine Molecular Medicine Molecular Biology |
| Zdroj: | ChemBioChem. 7:1577-1581 |
| ISSN: | 1439-4227 |
| DOI: | 10.1002/cbic.200600137 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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