Class Note for BME 510 at UA 2
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Date Created: 02/06/15
PERSPECTIVES INNOVATION A chemical toolkit for proteins an expanded genetic code Jlanmlng Xie and Peter C Schultz Abstract Recently a method to encode unnatural amino acids with diverse physicochemicai and biological properties genetically in bacteria yeast and mammalian cells was developed Over 30 unnatural amino acids have been coe transiationaiiy incorporated into proteins with high fidelity and efficiency using a unique codon and corresponding transfereRNA2aminoacyiitRNAesynthetase pair This provides a powerful tool for exploring protein structure and function in vitro and in vivo and for generating proteinswith new or enhanced properties Although the genetic codes of all known organisms specify the same 20 amino acids with the rare exceptions of selenocysteine1 and pyrrolysinez it is clear that numerous proteins require many cofactors and post translational modi cations to carry out their natural functions Therefore although a 207aminoeacid code might be suf cient for life it might not be optimal Consequently the development of a method that allows us to encode extra amino acids genetically might facilitate the evolution of proteins or even entire organisms with new or enhanced properties Moreover the ability to incorporate amino acids with de ned steric and electronic properties at unique sites in proteins will provide powerful new tools for exploring protein structure and function in vitro and in viva the ability to incorporate amino acids with de ned steric and electronic properties at unique sites in proteins will provide powerful new tools for exploring protein structure and function in vitro and in vivo Here we describe an approach that makes it possible for the rst time to add new amino acids to the genetic codes of both prokaryotic and eukaryotic organisms Over 30 unnatural amino acids 7 including those containing spectroscopic probes post translational modi cations metal chelators photoaf nity labels and other chemical moieties 7 have been selectively incorpoe rated into proteins with high delity and ef ciency in response to unique three and four base codons Methodology General considerations The incorporation of an unnatural amino acid at a de ned site in a protein directly in a living organism requires a unique transfereRNAzcodon pair a cor responding aminoacylatRNA synthetase and signi cant intracellular levels of the unnatural amino acid3i To ensure that the unnatural amino acid is incorporated uniquely at the site speci ed by its codon the tRNA must be constructed such that it is not recognized by the endogenous aminoacylatRNA synthetases of the host but functions ef ciently in translation an orthogonal tRNAi Moreover this tRNA must deliver the novel amino acid in response to a unique codon that does not encode any of the common 20 amino acids Another requirement for high delity is that the cognate aminoacylatRNA synthetase an orthogonal synthetase aminoacylates the orthogonal tRNA but does not aminoacylate any of the endogenous tRNAs Furthermore this synthetase must aminoacylate the tRNA with only the desired unnatural amino acid and not with the endogenous amino acids Similarly the unnatural amino acid cannot be a substrate for the endogenous synthetases if it is to be incorporated uniquely in response to its cognate codon Last the unnatural amino acid must be ef ciently transported into the cytoplasm when it is added to the growth medium or biosynthesized by the host and it must be stable in the presence of endogenous metabolic enzymes Several biochemical methods have pre viously been developed to insert unnatural amino acids into proteins However they require either in vitro protein synthesis or the stoichiometric use of chemically amino acylated tRNAsH which results in low protein yields or they result in the substitue tion of an unnatural amino acid which typically must be a close structural analogue of a common amino acid throughout the proteome or the partial incorporation of the unnatural amino acid in competition with endogenous amino acids The challenge is to develop a general method that makes it possible to incorporate a wide range of unnatural amino acids at any genetically speci ed site in the proteome with high translational delity and ef ciency Encoding unnatural amino acids in prokaryotes Initially the amber nonsense codon UAG was used to specify a novel amino acid in Escherichia coli because it is the least used stop codon and the presence of natural amber suppressors in some E coli strains does not signi cantly affect cellegrowth rates To obtain orthogonal tRNAaminoacylatRNAesynthetase pairs that uniquely encode extra amino acids in bace teria orthologues were taken from archaeai The rst such pair was derived from the tyrosylrtRNAtyrosylrtRNAesynthetase pair from Methanococcusjannaschii thRNAT l MjTyrRS thRNAT f has distinct synthetase recognition elements compared to bacterial tRNAs Flc la and the cognate synthetase MjTyrRS can be expressed ef ciently in E colii Also MjTyrRS has a minimalist anticodoneloopebinding domain which made it possible to alter the anticodon loop of thRNAT f to CUA with a minimal reduction in its affinity for the synthetasei Last M jTyrRS does not have an editing mechanism that could deacylate the unnatural amino aci 5 NATUREREVIEWS i MOLECULAR CELL BIOLOGY 2006 Namre Publishing Group VOLUME 7 iOCTOBER 2006 l115 3 3 3 h 93 A A A COH COH COH c c c c c 5 5 5 c3 C 0 Pc c Pu A PC C c c u A c c c c c c c c u c c c c c c c c c c c c c A c c A c c c c A ccA AUCC cccccU CccA HUCC cuuccU A DocA AUCC cccccU A ccA AUCC cccocU U we ccuccu CA c we CAACCU c Acoc coccc cc we ccuccu CA CCUACAAcc cU u cc AAccc A u cD AAccc c cD u cc ACAA cC c u chC A cc A DD ccACA ccAuC c c c c A u c c c c A u A u c c E C C C i C E C u A u u u A c A c A c A u c u A u A u A U A Mannaschzz RNA Y E coll tRNA quot 5 cerevzsme RNAW An orthogonal MJtRNAELAm Ecolz c MJtRNAEAjA MT RS Pos veselection Sumvorscomam M yrRsznantsthat TAG lbw gt can chargetheunnaturalorany natural 0155 Cm WY viz r aturalammo aminoacwdontotheorthogonal RNA H70 vCHloramphemcol Deswred M yrRS vanant Undeswred M yrRSvanant L65 of m We charges unnatural amino acid charges natural amino acwd sele L162 V32 Transform WRNA gg MJtRNA gA D158 Deswred TAG TACTAC Ba m ase Unnatural ammo sad approach was developed that involved generating a large library 109 mutants of synthetase activeesite mutants19gt20 Pic lb followed by a combination of positive and negative selections to identify a synthetase with the desired speci city Pic ici The positive selection was based on resistance to chloramphenicol which in the presence ofthe unnatural amino acid and MjTyrRS was conferred by the suppression of an amber mutation at a permissive site in the chloramphenicol acetyltransferase gene The negative selection used the toxic barnase gene with amber mutations at permissive sites and was carried out in the absence ofthe unnatural amino acid Only MjTyrRS variants that could acylate the orthogonal thRNAglJAwith the unnatural amino acid and not with the endogenous amino acids could survive both selectionsi This selection scheme and more recent variants have been used to develop MjTyrRS mutants that are capable of selec7 tively inserting over 30 unnatural amino acids into proteins in E coli in response to the amber codon3i Typically 5710 mgl l of an unnaturalfaminoeacidecontaining pro tein can be obtained from minimal media with translational fidelities of over 99 Recently the system was optimized which has made it possible to produce approxi7 mately 500 mg l 1 of proteins that contain unnatural amino acids in bacteria using highedensity fermentationZ i Meanwhile further orthogonal suppressoretRNA aminoacylitRNAesynthetase pairs have been generatedZHS which increase the structural diversity and number of unnatue ral amino acids that can be incorporated into proteins using this method Moreover we have shown that it is possible to add an engineered pathway for the biosynthesis of an unnatural amino acid peaminophenyle alanine into E coli to generate an autonomous 217aminoeacid bacteriumzsi Encoding unnatural amino acids in eukaryotes A similar strategy has been used to generate nonsenseisuppressore tRNAaminoacylitRNAesynthetase pairs in Saccharomyces cerevisiae from E coli orthologueszmi In this case a positive and negative selection scheme was developed that used the transcriptional activator 11 I with two amber mutations at permissive sitesi Suppression ofthese amber codons led to the production of full length Gal4 which in turn drove the transcription of positive or negative selection reporters This straightforward selection scheme together with orthogonaletRNAzaminoe acylrtRNAesynthetase pairs 7 including E coli tRNAE AzTyrRS and tRNA g JA leucylitRNAesynthetase pairs 7 has allowed us to incorporate over 15 unnatural amino acids into proteins in S cerevisiae Expression levels of up to 75 mg l 1 have been obtained with greater than 98 f1delitiesi More recently the mutant aminoacyli tRNA synthetases that were evolved in S cerevisiae to accept unnatural amino acids have been used together with a Bacillus stearothermophilus amber suppressor tRNAllJA to selectively insert various unnate ural amino acids into proteins in mam malian cells in response to nonsense codons W Liu and RGiSi unpublished results Also Yokoyama and coworkers screened a collection of designed activeesite variants of E coli TyrRS in a wheategerm translation system and discovered a mutant synthetase that uses 37iodotyrosine more effectively than Tyrlgi This mutant synthetase was later used with B stearothermophilus tRNAl f A to incorporate 37iodotyrosine into proteins in mammalian cells3 i Similarly we used a mutant orthogonal Bacillus subtilis tRNAE A tryptophanylrtRNAasynthetase pair to introduce the redoxeactive crosslinking agent Sehydroxytryptophan selectively into proteins in response to the UGA opal codon3 i However it remains a challenge to produce large quantities of proteins containing unnatural amino acids in mammalian cells we have shown that it is possible to add an engineered pathway for the biosynthesis of an unnatural amino acid into E coli to generate an autonomous 21aminoacid bacterium Further codonsfor unnatural amino acids To incorporate two or more distinct unnatural amino acids into a single protein simultaneously further unique codons other than the amber and opal nonsense codons are needed It should be possible to use quadruplet codons and cognate sup pressor tRNAs with expanded anticodon loops to specify unnatural amino acidsm Recently we showed that an orthogonal tRNAgnglysylrtRNAisynthetase pair from Pyrococcus horikosln39i could be used to incorporate homoglutamine selectively into proteins in E coli in response to the PERSPECTIVES quadruplet codon AGGAi The combina7 tion ofamber and AGGA suppressions allowed the simultaneous incorporation of two unnatural amino acids at distinct sites in a single protein23i An alternative approach to generate further codons involves eliminating degenerate nonsense codons and codonrtRNA pairs from the E coli genome avoiding competition with stop or coding codonsi We are cur rently evaluating methods for the efficient construction ofa partially codonedeleted E coli genomei A remaining challenge is to define the role ofcontext effects in sup pression efficiency the efficiency with which nonsense codons can be suppressed can be affected by the characteristics of adjacent codons and to identify genomic mutations for example in the ribosome tRNAs and elongation factors that improve suppression efficiency An expanded genetic code The above methodology has been success fully used to add a large number of diverse unnatural amino acids to the genetic codes of E col 139 S cerevisiae and mammalian cells Many of these unnatural amino acids have novel properties that are useful for various biochemical and cellular studies of protein structure and function Pic 2L Chemically reactive groups A potential general strategy for selective protein modie fication involves the siteespecific incorporae tion of unnatural amino acids with novel reactivity into proteins which can subse7 quently be derivatized with high efficiency and selectivity Indeed several unnatural amino acids with reactive groups includ ing ketone azide acetylene and thioester groups have been genetically encoded in E coli and S cerevz39siaem 9 178 in HQ 2 These chemistries have been used to modify proteins selectively under mild con ditions with a number of uorophore35gt35gt 0 tags and other exogenous reagentsi In one example a series of uorescent dyes were selectively introduced at a unique site in an meacetylphenylalanine mutant of the membrane protein Am B in E coll In a second example a mutant human growth hormone was siteespecifically modified with polyethylene glycol PEG with a high yield to create a protein that retained wildetype activity but that had a consider ably improved halfelife in serum Hi Cho and T Daniel unpublished results This work is currently being extended to other therapeutic proteins as well as to the gen eration of homodimeric and heterodimeric NATUREREVIEWS l MOLECULAR CELL BIOLOGY 2006 Namre Publishing Group VOLUME 7 lOCTOBER 2006 i111 O O O OH O NH2 5 0 Se 0 I HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH 1 2 3 4 5 6 7 o HTN Q N ON ON O N 3 CF 2 Z N f 10 HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH s 9 1o 11 12 13 14 OH O OH HO O 0 93 OH O OH N NO HO 0 0 CH3 2 O O AcHN HO O O NH NHAc HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH 15 15 17 13 19 20 21 HO OH HHZ Br 0 CN OH OH CH NH CN HZN COOH HZ SH COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH 22 23 24 25 25 27 28 0 SH 0 N O HHZ N HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH HZN COOH 29 3o 31 32 33 34 35 dimer interface could be crosslinked in the cytoplasm of E coli cells with a greater than 50 yield This amino acid was recently used as a substitute for residues in the central pore site ofi Ilpli a ringcforming AAA ATPases associated with various cellular activities protein that rescues proteins from aggregated states The resulting mutants were shown to photocrosslink to the peptide substrate which provided direct physical evidence for close contacts between the ClpB pore site and substrates i Yokoyama and co sworkers also showed that pcbenzoylphenylc alanine can be selectively incorporated into human ililil growthcfactorcreceptorcbound proteinc2 in Chinese hamster ovary cells and crosslinked to the epidermalcgrowthcfactor receptorAT These amino acids should be use ful as probes of protein interactions protein structure and protein dynamics in vitro and in vivo and they should also be helpful in identifying receptors for orphan ligands Photocaged Cys Ser and Tyr amino acids 11713 respectively in Pic 2 have also been sitecspeci cally introduced into proteins using this methodology The sidecchain hydroxy or thiol groups of these amino acids are blocked by substituted nitrobenzyl groups that can be cleaved on irradiation with 365nm light in vitro or in viioi In one exams ple the activecsite Cys of the procapoptotic cysteine protease caspasec3 was substituted with nitrobenzyl Cys to create an inactive protein that could be photoactivated with greater than 70 ef ciency In a related experiment the photochromic 9 amino acid pcazophenylcphenylalanine 14 in Pic 2 was sitecspeci cally introduced into the cyclic AMPcbinding site of the E coli transcription factor catabolite activator protein CAP Irradiation of this amino acid with 334cnm light predominantly converts the trans form of the amino acid to the 5139s formi Because the two isomers differ signi cantly in structure and dipole the cis and trans mutants have dif ferent af nities for cAMP and as a result the affinity of this mutant CAP for its promoter can be photoregulatedc Similarly it should be possible to photomodulate the activity of other enzymes for example kinases and phosc phatases receptors and transcription factors Postctrunslutionul modifications The dif culties that are associated with the gen eration of selectively glycosylated proteins have hindered our understanding of the biological roles of glycosylation and have also made the production of therapeutically useful glycoproteins challengings i To begin to provide a general approach for the syn thesis of structurallycdefmed glycoproteins PERSPECTIVES o myoglobineTAc4 uorescence emission 450 nm E MyoglobineTAGw uorescence emission 450 nm myoglobineTAczi molar ellipticity 222 nm Q myoglobineTAcw molar ellipticity 222 nm Fraction folded 0 l l l l l 0 1 Z 3 4 5 Urea concentration M Figure 3 l The sitespecific incorporation of a coumarinderived fluorescent amino acid into myoglobin as a probe of protein conformational changes An orthogonalMethanococcusjanc naschii tyrosylitransfercRNA WIjtRNAEggztyrosylitRNAcsynthetase pair was identified that specifically incorporates a coumarincderived amino acid 18 in HQ 2 into proteins in responsetothe amberTAG codoni Because coumarin fluorescence is sensitive to solvent polarity its fluorescence intensityshould correlate with any local unfolding ofthe protein that occurs in close proximitytothe mutated position To illustratethisthe coumarincderived amino acid was incorporated at position 4 or 37 ofsperm whale myoglobin myoglobiniTAG4 or myoglobiniTAG3 7 respectivelyi The resulting mutants were unfolded by urea and this was monitored by measuring the fluorescence intensity ofthe incorporated cou marinylamino acid and by circular dichroism CD In the presence of 5 M urea fully unfolded state both ofthe myoglobin mutants showed a 30 increase in their fluorescence signalcompared tothat at 0 M urea fully folded state which indicatesthat the fluorescence intensity of cou ma rin correlates with protein conformational changes The fraction folded was calculated by normalizing the fluores cence signalorthe molarellipticity which is an experimental parameter for CD at 5 M urea tothe fully unfolded state On moving from 0 M to Z M ureathe fluorescence intensity of myoglobiniTAG4 increased by 25 and remained roughly atthis levelfrom Z M to 5 M urea which indicatesthatthis region ofthe protein is disordered By contrast myoglobiniTAG37 showed little change in its fluores cence intensity at Z M urea but underwent a similarfluorescence increase 25at 3 M urea This is consistent with N MR data which indicate that helices A and B of myoglobin are largely disordered when the urea concentration is higherthan 212 M helixA contains residue 4 whereas helices C D and F unfold when the urea concentration is higherthan 310 M helixC contains residue 37i It therefore seems thatthe coumarinylamino acid is a sitecspecific probe of protein conformationalchangesiThe CD measurements produced virtually identical unfolding curves for myoglobiniTAG4 and myoglobini TAG37 which is consistent with the efficacy of CD in reporting global conformational changes that are averaged overthe entire structure mutant synthetases have been evolved that sitecspeci cally incorporate Bchacetylc glucosaminecOcserine BcGlcNAccSer 15 in Pic 2 into proteins in E coli It was shown that a BcGlcNAccSerccontaining mutant myoglobin that was generated by this method could be further modified by galacc tosyltransferases to produce more complex saccharides A similar approach was used to introduce achacetylgalactosaminecOcthrec onine 16 in Pic 2 selectively into proteins and is currently being extended to a number of other on and Nclinked sugars Reversible protein phosphorylation princ cipally on Ser Thr or Tyr residues is crucial in the regulation of signalctransduction pathways Consequently the generation of selectively phosphorylated proteins or stable analogues of phosphoproteins would be usefuls h As a rst step we have selectively incorporated a noncphosphorusc containing analogue of phosphotyrosine 7 pcarboxymethyLLsphenylalanine 17 in Pic 2 7 into proteins in E coli When this unnatural amino acid is used to replace Tyr701 in human l39l 1 signal transducer and activator of transcriptioncl the result ing protein dimerizes and binds to the same DNA sequence as Tyr701cphosphorylated STATl REF 551 Because this amino acid has better cellular membrane permeability than phosphotyrosine and is resistant to protein tyrosine phosphatases it should be useful in the generation of other stable phosphoprotein analogues orpeptidecbased inhibitors for SHZ Srcchomologch domains NATUREREVIEWS l MOLECULAR CELL BIOLOGY VOLUME 7 lOCTOBER 2006 1119 2006 Namre Publishing Group there is a great deal of structural plasticity in the conformations of the aminoeacid side chains and the protein backbone in the aminoeacidebinding sites of these amino acylrtRNA synthetases55gt66 Flc 4i It should therefore be possible to add other novel amino acids to the genetic code including spin labels electronetransfer mediators neareinfrared probes and longechain alkanes as well as building blocks with altered backbones such as OLthdroxy acids and Nealkyl amino acids Conclusions The approach described above has proven remarkably effective in allowing us to add a large number of structurally diverse amino acids to the genetic codes ofboth prokaryotic and eukaryotic organisms The ability to encode unnatural amino acids genetically should provide powerful probes of protein structure and function in vitro and in viioi It might also allow the design or evolution of proteins with novel proper ties Possible examples include the rational design of glycosylated or PEGylated therapeutic proteins with improved pharmacological properties uorescent proteins that function as sensors of small molecules and proteinrprotein interace tions in cells and proteins with activities that can be photoregulated in viioi We might also be able to select for peptides and proteins that have enhanced function using libraries of unnaturaleaminoeacid mutantsi For example we recently showed that it is possible to incorporate unnatural amino acids into phageedisplayed pep tides and a peptide with an enhanced affinity for streptavidin was isolated and found to contain an unnatural amino acid It should also be possible to incorporate noneaminoeacid building blocks into proteins or perhaps to create biopolymers with entirely unnatural backbones as well as to generate multicellular organisms that contain unnatural amino acids Last the capability to add novel amino acids to the genetic code of organisms should allow us to test experimentally whether organisms with genetic codes that contain more than twenty aminoeacid building blocks have an evolutionary advantagei Jianming Xie and Peter C Schultz are at the Department of Chemisz and Skaggs Institute for Chemical Biology the Scripps Research Insu39tute l 0550 North Torrey Pines Road La Jolla California 9203 7 USA Correspondence to 265 esmail schultzscripps edu doll l058llrm2005 Publlslled onllne 25 August 2006 20 22 25 24 25 BocxA etol Selenocysteine tne 21stamino acid Mol Microoiol 5515520 ll99l Srinivasanc JamesC M BlKlzycklJ A Pyrfolyslrle encoded by UAC in arcnaea cnargingofa UAcr decoding specialized tRNA Science 296 145971 462 2002 Wang L ampSchult1P c Expandingtnegenetic code AngeW Chem Int Edh Eng 44 34766 2004 Cornisnv w Mendel D ampSchult1P c Probing protein structure and function with an expanded genetic code AngeW Chem Int Edh Eng 34 217633 1995 BalrlJ DClabeC c DixTACnamberlinA R 6i DialaE S Biosyntneticsitespecificincorporationofa rlorlrrlatufal aminoacid intoa polypeptide J Am Chem Soc 1118olseaol4ll989 Beene D L Dougnerty D A 6i Lester H A Unnatural amino acid mumgenesls in mapping ion channel function Curr 0pm Neurooiol 13 2647270 2003 Hortinc ampBolmel Applications ofamino acid analogs for studying co and posttranslational modi cationsofproteins MeoiodsEhzymol 96 7777784 ll 983 Eurter R Expansion oftne genetic code slterdllected pr uomrpherlylalarllrle incorporation inEscherichio coli Protein Sci 7 41 97426 1998 Doring v etol Enlargingtne aminoacid set of Escherichia coli by in ltration oftne valine coding patnway Science 292 5017504 2001 Klfsherlbaum KCarrlcol S BlTlrfeH D A Biosynthesis of proteins incorporating a versatile set of phenylalanine analogues Chemoiochem 3 2357237 2002 Benzer S 6i cnampe S P Acnange from nonsense to sense in the genetic code ProC NodAcod Sci USA 43 llllrrllzl 1962 Caren A 6i Siddidi o Suppression ofmutations in the alkaline phosphatase structural cistron ofE coli ProC NotlAcod Sci USA43 112171127 1962 Wang L MaglieryT J Liu D R ampSchult1P c A new functional suppressortRHAaminoacyltfeHA synthemse pairfor the in vivo incorporation of unnatural aminoacids into proteins J Am Chem Soc 122 SOlOrSOll 2000 Eecnter P RudlrlgerrThlrlorlJ Tukalo M 6i ciege R Malor tyrosine identity determinants in Methohococcus ohhoschii and Socchoromyces cerevisioe tRNA are conserved but expressed differently Eur J Biochem 2687617767 21ml Steer B A ampScnimmel P Malorantlcodonrblndlng region missing from an arcnaebacterial tRNA synthemse J Biol Chem 274 35601735606 1999 Jakubowskl H BlColdmarl E Editingoferrors in selection ofamino acids for protein synthesis Microoiol Rev 56 4127429 1992 Wang L ampSchult1P c Ageneral approacn fortne generation oforthogorlaltRNAs Chem Biol 3 8837890 21ml Wang L Brocx A Herbericn B 6i Schultz P 0 Expanding the genetic code ofEscherichio coli Science 292 4987500 21ml Kobayashl T etol Structural basis forortnogonal tRNAspeclflcltles oftyfosylstRNAsyrlthemses for genetic code expansion NotureSouct Biol 10 4257432 2003 znangvWang LSchult1 P c BlWllsorl l A Crystal structures ofapowlldrtypeM ohhoschiityrosyltfeHA synthemse TyrRS and an engineered TyrRS speci c for Omethylrutyroslrle Protein Sci 14 134071349 2005 Ryu v 6i Schultz P 0 Ef cient incorporation of unnatural amino acids into proteins in Escherichio coli Noture Methods 3 2637265 20061 AndersonJ C ampSchult1P c Adapmtlorlofarl ortnogonal arcnaeal leucyltRHAand syntnetase pair for fourrbase amber and opal suppression Biochemistry 42 959879608 2003 AndersonJ C etol An expanded genetic codewitna functionalduadrupletcodon ProC NotlAcod Sci USA 10175667757l 2004 KowalA K Kohrer C BlRalBharldafy u L Twenty firstaminoacyltfeHAsyntneaseesuppressortfeHA pairs for possible use in sitespecific incorporation of amino acid analogues into proteins in euxaryotesand in eubacteria ProC NotlAcod Sci USA 93 226872273 21ml Santoro S WArldelsorlJ C Laxsnmanv ampScnuliz P c An arcnaebacteriaderived gluamyltfeHA synthemse and tRNApalrfor unnatural aminoacid mumgenesls of proteins in Escherichio coli Nucleic Acids Res 31670076709 2003 26 27 28 29 SO 52 55 54 55 56 57 58 59 40 42 45 44 45 46 47 4s 49 50 PERSPECTIVES Menl R A etol Generation ofa bacteriumwitn a 21 amino acid genetic code J Am Chem Soc 125 9357939 2003 cninl w etol An expanded euxaryotic genetic code Science 301 9647967 2003 Wu N Deiters A CroppT A Klrlg D 6i Schultz P c Ageneticallyencoded pnotocaged aminoacid J Am Chem Soc 126 1430644307 2004 Klga D etol An engineered Escherichio colityrosyle tRNAsyrlthemse forslterspeclflc incorporation ofan unnatural amino aCid into protelrls in eukaryotlc translation and itsapplication in awneat germ cellfree system ProC NotlAcod Sci USA 99 971579720 2002 Saxamoto K etol Sitespecific incorporation ofan unnatural amino acid into proteins in mammalian cells NucleicAcids Res 30 469274699 2002 znang z etol Selective incorporation of Srhydfoxytfyptopharl into proteins in mammalian cells ProC NotlAcod Sci USA 101 888278887 2004 HonsaxaTAsnizuxavTaira H Muraxami H 6i Sisido M lncorporation of nonnatural amino acids into proteins by usingvariousfourbase codons in an Escherichio coliih vioo translation system Biochemistry 40 1106041064 2001 AndersonJ C MagllefyTJ ampSchult1P 0 Exploring the limits ofcodon and anticodon size Chem Biol 9 2377244 2002 EeinsteinS l ampAltmanS Contexteffects on nonsense codon suppression in Escherichio coli Genetics 33 2017219 1978 Wang Lznangz BrocxA ampSchult1P 0 Addition oftne xeto functional group to the genetic code of Escherichio coli ProC Nod Acod Sci USA 100 56761 2003 znang 2 et ol A new strategy for the
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