Expanding the genetic code.

Recently, a general method was developed that makes it possible to genetically encode unnatural amino acids with diverse physical, chemical, or biological properties in Escherichia coli, yeast, and mammalian cells. More than 30 unnatural amino acids have been incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding tRNA/aminoacyl-tRNA synthetase pair. These include fluorescent, glycosylated, metal-ion-binding, and redox-active amino acids, as well as amino acids with unique chemical and photochemical reactivity. This methodology provides a powerful tool both for exploring protein structure and function in vitro and in vivo and for generating proteins with new or enhanced properties.

[1]  J. Miller,et al.  Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity. , 1990, Journal of molecular biology.

[2]  J. Wong,et al.  Evolutionary relationship between Halobacterium cutirubrum and eukaryotes determined by use of aminoacyl-tRNA synthetases as phylogenetic probes. , 1980, Canadian journal of biochemistry.

[3]  P G Schultz,et al.  Expanding the Genetic Code of Escherichia coli , 2001, Science.

[4]  Jacob M Hooker,et al.  Interior surface modification of bacteriophage MS2. , 2004, Journal of the American Chemical Society.

[5]  Andrew B. Martin,et al.  Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  P. Schimmel,et al.  A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase , 1990, Molecular and cellular biology.

[7]  P. Dawson,et al.  Synthesis of native proteins by chemical ligation. , 2000, Annual review of biochemistry.

[8]  Andrew B. Martin,et al.  Generation of a bacterium with a 21 amino acid genetic code. , 2003, Journal of the American Chemical Society.

[9]  B. Crane,et al.  Structure and activity of an aminoacyl-tRNA synthetase that charges tRNA with nitro-tryptophan , 2005, Nature Structural &Molecular Biology.

[10]  P. Schultz,et al.  Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression. , 2003, Biochemistry.

[11]  W A Hendrickson,et al.  Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three‐dimensional structure. , 1990, The EMBO journal.

[12]  A F Carne,et al.  Chemical modification of proteins. , 1994, Methods in molecular biology.

[13]  P. Schultz,et al.  The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination , 2004, Nature Biotechnology.

[14]  Peter G Schultz,et al.  An Expanded Eukaryotic Genetic Code , 2003, Science.

[15]  H. Hennecke,et al.  Relaxing the substrate specificity of an aminoacyl‐tRNA synthetase allows in vitro and in vivo synthesis of proteins containing unnatural amino acids , 1995, FEBS letters.

[16]  P. Schultz,et al.  A general approach for the generation of orthogonal tRNAs. , 2001, Chemistry & biology.

[17]  S. Yokoyama,et al.  Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells. , 2002, Nucleic acids research.

[18]  Shigeyuki Yokoyama,et al.  Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion , 2003, Nature Structural Biology.

[19]  P. Schultz,et al.  Site-specific incorporation of a redox-active amino acid into proteins. , 2003, Journal of the American Chemical Society.

[20]  Joseph A. Krzycki,et al.  Pyrrolysine Encoded by UAG in Archaea: Charging of a UAG-Decoding Specialized tRNA , 2002, Science.

[21]  A. Weiner,et al.  Crystal Structures of the Bacillus stearothermophilus CCA-Adding Enzyme and Its Complexes with ATP or CTP , 2002, Cell.

[22]  David R. Liu,et al.  A New Functional Suppressor tRNA/ Aminoacyl-tRNA Synthetase Pair for the in Vivo Incorporation of Unnatural Amino Acids into Proteins , 2000 .

[23]  Daisuke Kiga,et al.  An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Probing Protein Structure and Function with an Expanded Genetic Code , 1995 .

[25]  Peter G Schultz,et al.  A genetically encoded photocaged amino acid. , 2004, Journal of the American Chemical Society.

[26]  P. Schultz,et al.  Site-specific PEGylation of proteins containing unnatural amino acids. , 2004, Bioorganic & medicinal chemistry letters.

[27]  T. C. Evans,et al.  Intein-mediated protein ligation: harnessing nature's escape artists , 1999 .

[28]  Peter G Schultz,et al.  Unnatural amino acid mutagenesis of green fluorescent protein. , 2003, The Journal of organic chemistry.

[29]  Bruce Stillman,et al.  Cold Spring Harbor Laboratory , 1995, Current Biology.

[30]  G. Hortin,et al.  Applications of amino acid analogs for studying co- and posttranslational modifications of proteins. , 1983, Methods in enzymology.

[31]  Simon J North,et al.  N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. , 2002, Science.

[32]  P. Schultz,et al.  The incorporation of a photoisomerizable amino acid into proteins in E. coli. , 2006, Journal of the American Chemical Society.

[33]  Andrew B. Martin,et al.  Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. , 2002, Journal of the American Chemical Society.

[34]  D. Kim,et al.  Construction, expression, and function of a new yeast amber suppressor, tRNATrpA. , 1988, The Journal of biological chemistry.

[35]  B. P. Doctor,et al.  SPECIES SPECIFICITY OF AMINO ACID ACCEPTOR RIBONUCLEIC ACID AND AMINOACYL SOLUBLE RIBONUCLEIC ACID SYNTHETASES. , 1963, The Journal of biological chemistry.

[36]  M. Yarus,et al.  Reading frame selection and transfer RNA anticodon loop stacking. , 1987, Science.

[37]  P. Schultz,et al.  Adding L-3-(2-Naphthyl)alanine to the genetic code of E. coli. , 2002, Journal of the American Chemical Society.

[38]  P. Schultz,et al.  An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli. , 2003, Nucleic acids research.

[39]  P. Schultz,et al.  Expanding the genetic code , 2022, Protein science : a publication of the Protein Society.

[40]  Peter G Schultz,et al.  An expanded genetic code with a functional quadruplet codon. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[41]  F. Dahlquist,et al.  Biosynthetic Incorporation of 15N and 13C for Assignment and Interpretation of Nuclear Magnetic Resonance Spectra of Proteins , 1990, Quarterly Reviews of Biophysics.

[42]  S. Kuersten,et al.  The role of exportin‐t in selective nuclear export of mature tRNAs , 1998, The EMBO journal.

[43]  Benjamin G Davis,et al.  Synthesis of glycoproteins. , 2002, Chemical reviews.

[44]  P. Schimmel,et al.  An Escherichia coli tyrosine transfer RNA is a leucine-specific transfer RNA in the yeast Saccharomyces cerevisiae. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[45]  P G Schultz,et al.  A general method for site-specific incorporation of unnatural amino acids into proteins. , 1989, Science.

[46]  J. Broach,et al.  The Molecular biology of the yeast Saccharomyces : metabolism and gene expression , 1982 .

[47]  Teresa Mitchell,et al.  Production of Complex Human Glycoproteins in Yeast , 2003, Science.

[48]  P. Schultz,et al.  A New Orthogonal Suppressor tRNA/Aminoacyl‐tRNA Synthetase Pair for Evolving an Organism with an Expanded Genetic Code , 2000 .

[49]  C. Glabe,et al.  Biosynthetic site-specific incorporation of a non-natural amino acid into a polypeptide , 1989 .

[50]  Raymond A. Dwek,et al.  Glycobiology: Toward Understanding the Function of Sugars. , 1996, Chemical reviews.

[51]  Chemical synthesis of peptides and proteins. , 1988, Annual review of biochemistry.

[52]  P. Schultz,et al.  Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo. , 2001, Bioorganic & medicinal chemistry.

[53]  Peter G. Schultz,et al.  A New Strategy for the Synthesis of Glycoproteins , 2004, Science.

[54]  P. Schultz,et al.  Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[55]  Chi‐Huey Wong,et al.  Toward Automated Synthesis of Oligosaccharides and Glycoproteins , 2001, Science.

[56]  L. Bossi,et al.  Four-base codons ACCA, ACCU and ACCC are recognized by frameshift suppressor sufJ , 1981, Cell.

[57]  Peter G Schultz,et al.  Exploring the limits of codon and anticodon size. , 2002, Chemistry & biology.

[58]  M. O’Connor Insertions in the anticodon loop of tRNA1Gln(sufG) and tRNA(Lys) promote quadruplet decoding of CAAA. , 2002, Nucleic acids research.

[59]  Lei Wang,et al.  Crystal structures of apo wild‐type M. jannaschii tyrosyl‐tRNA synthetase (TyrRS) and an engineered TyrRS specific for O‐methyl‐L‐tyrosine , 2005, Protein science : a publication of the Protein Society.

[60]  L. Craig,et al.  Benzophenone triplet: a new photochemical probe of biological ligand-receptor interactions. , 1973, Nature: New biology.

[61]  P. Schultz,et al.  Addition of the keto functional group to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[62]  D. Söll,et al.  Aminoacyl-tRNA synthesis. , 2000, Annual review of biochemistry.

[63]  P. Schimmel,et al.  Major Anticodon-binding Region Missing from an Archaebacterial tRNA Synthetase* , 1999, The Journal of Biological Chemistry.

[64]  U. RajBhandary,et al.  Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[65]  Peter G Schultz,et al.  A method for the generation of glycoprotein mimetics. , 2003, Journal of the American Chemical Society.

[66]  A. Böck,et al.  Selenocysteine: the 21st amino acid , 1991, Molecular microbiology.

[67]  P. Schultz,et al.  In Vivo Photocrosslinking with Unnatural Amino Acid Mutagenesis , 2002, Chembiochem : a European journal of chemical biology.

[68]  M. Birnstiel,et al.  Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements , 1981, Nature.

[69]  Peter G Schultz,et al.  A phage display system with unnatural amino acids. , 2004, Journal of the American Chemical Society.

[70]  P G Schultz,et al.  Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of “shifty” four-base codons with a library approach in Escherichia coli , 2001, Journal of Molecular Biology.

[71]  M. Sisido,et al.  Efficient Incorporation of Nonnatural Amino Acids with Large Aromatic Groups into Streptavidin in In Vitro Protein Synthesizing Systems , 1999 .

[72]  P. Marlière,et al.  Enlarging the Amino Acid Set of Escherichia coli by Infiltration of the Valine Coding Pathway , 2001, Science.

[73]  T. Muir,et al.  Protein semi-synthesis in living cells. , 2003, Journal of the American Chemical Society.

[74]  Shigeyuki Yokoyama,et al.  Structural snapshots of the KMSKS loop rearrangement for amino acid activation by bacterial tyrosyl-tRNA synthetase. , 2005, Journal of molecular biology.

[75]  H. Lester,et al.  Unnatural amino acid mutagenesis in mapping ion channel function , 2003, Current Opinion in Neurobiology.

[76]  P. Sharp,et al.  Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes , 1982, Cell.

[77]  Brian A. Smith,et al.  A new strategy for the site-specific modification of proteins in vivo. , 2003, Biochemistry.

[78]  P. Schultz,et al.  Characterization of an 'orthogonal' suppressor tRNA derived from E. coli tRNA2(Gln). , 1997, Chemistry & biology.

[79]  P. Schultz,et al.  Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. , 2003, Journal of the American Chemical Society.

[80]  P. Schultz,et al.  Selective Staudinger Modification of Proteins Containing p‐Azidophenylalanine , 2005, Chembiochem : a European journal of chemical biology.

[81]  P. Schultz,et al.  Site‐Specific in vivo Labeling of Proteins for NMR Studies , 2005, Chembiochem : a European journal of chemical biology.

[82]  S. Yokoyama,et al.  Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid , 2005, Nature Methods.

[83]  P. Schultz,et al.  Structural characterization of a p-acetylphenylalanyl aminoacyl-tRNA synthetase. , 2005, Journal of the American Chemical Society.

[84]  H. Drabkin,et al.  Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene , 1996, Molecular and cellular biology.

[85]  O. Siddiqi,et al.  Suppression of mutations in the alkaline phosphatase structural cistron of E. coli. , 1962, Proceedings of the National Academy of Sciences of the United States of America.

[86]  P. Schultz,et al.  Progress toward the evolution of an organism with an expanded genetic code. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[87]  P. Schultz,et al.  Expanding the genetic code. , 2002, Chemical communications.

[88]  R. Giegé,et al.  Major tyrosine identity determinants in Methanococcus jannaschii and Saccharomyces cerevisiae tRNA(Tyr) are conserved but expressed differently. , 2001, European journal of biochemistry.

[89]  S. Benzer,et al.  A change from nonsense to sense in the genetic code. , 1962, Proceedings of the National Academy of Sciences of the United States of America.

[90]  S. Yokoyama,et al.  Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[91]  Yi Tang,et al.  Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo. , 2002, Biochemistry.

[92]  P. Schultz,et al.  An efficient system for the evolution of aminoacyl-tRNA synthetase specificity , 2002, Nature Biotechnology.