Relaxing the substrate specificity of an aminoacyl-tRNA synthetase allows in vitro and in vivo synthesis of proteins containing unnatural amino acids.
It has previously been demonstrated that the unnatural amino acid p-Cl-phenylalanine can be attached to tRNA(Phe) by a modified phenylalanyl-tRNA synthetase with relaxed amino acid substrate specificity. We show that this modification to the translational machinery of Escherichia coli is the only requirement for the incorporation of either p-Cl- or p-Br-phenylalanine into full-length luciferase in vitro. The incorporation of p-Cl-phenylalanine was also demonstrated in vivo using a suitably modified host strain. These results represent the first description of the incorporation into a protein in vivo of an unnatural amino acid which is normally rejected by the cellular translational machinery.
Ibba
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Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH Zentrum, Zürich, Switzerland.
Hennecke
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D018833:Chaperonins / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D010134:Fenclonine / Q000378:metabolism; D007163:Immunosorbent Techniques; D008156:Luciferases / Q000096:biosynthesis / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012486:Salmonella typhimurium; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
C060302:4-bromophenylalanine; D010649:Phenylalanine; D008156:Luciferases; D018833:Chaperonins; D010652:Phenylalanine-tRNA Ligase; D010134:Fenclonine