Strategies for in vitro and in vivo translation with non-natural amino acids.
The use of site-directed mutagenesis (Smith, 1985) to replace amino acids at any chosen position in a protein, coupled with advances in analytical procedures, has greatly advanced our understanding of biological structure-function relationships in recent years. The only limitation of conventional site-directed mutagenesis is that substitutions are restricted to the 20 naturally occurring amino acids. However, the discovery of a 21st amino acid, selenocysteine, and the development of novel in vitro translation techniques have demonstrated that considerably more site-specific replacements are possible during protein engineering. These techniques have already found a wide range of applications and have shown that the translational machinery is able to accommodate an enormously divergent range of aminoacylated tRNAs. Although these techniques are mainly restricted to in vitro systems, recent progress in our understanding of aminoacyl-tRNA synthetase-catalyzed tRNA charging suggests that it may ultimately be possible to extend this technique to growing cells.
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Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009239:N-Formylmethionine / Q000737:chemistry; D014176:Protein Biosynthesis; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D017279:Selenocysteine / Q000235:genetics; D051140:Selenoproteins
D000596:Amino Acids; D003062:Codon; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D051140:Selenoproteins; D017279:Selenocysteine; D009239:N-Formylmethionine; D000604:Amino Acyl-tRNA Synthetases