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Peter G Schultz - One of the best experts on this subject based on the ideXlab platform.
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an archaebacteria derived glutamyl trna synthetase and trna Pair for unnatural amino acid mutagenesis of proteins in escherichia coli
Nucleic Acids Research, 2003Co-Authors: Stephen W Santoro, Christopher J Anderson, Vishva Lakshman, Peter G SchultzAbstract:The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA Pair that is ‘Orthogonal’, meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the Orthogonal synthetase is then modified to charge the corresponding Orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an Orthogonal glutamic acid synthetase and tRNA Pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNAGlu sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new Orthogonal Pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the Orthogonal tyrosine Pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid Orthogonal Pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.
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a new functional suppressor trna aminoacyl trna synthetase Pair for the in vivo incorporation of unnatural amino acids into proteins
Journal of the American Chemical Society, 2000Co-Authors: Lei Wang, Thomas J Magliery, Peter G SchultzAbstract:General methods for selectively incorporating unnatural amino acids into proteins in vivo, directly from the growth media, would greatly expand our ability to manipulate protein structure and function.1 For example, the ability to place fluorophores selectively into proteins in vivo would provide powerful tools for cell biology, or the ability to generate large quantities of proteins with metal binding or keto amino acids might lead to proteins with enhanced physical or catalytic properties. Our approach involves the generation of a suppressor tRNA/aminoacyl-tRNA synthetase (tRNACUA/aaRS) Pair that is Orthogonal to Escherichia coli endogenous tRNA/synthetase Pairs; that is, the Orthogonal tRNA is not a substrate for any endogenous synthetases and the Orthogonal synthetase does not recognize any endogenous tRNAs.2,3 The specificity of this synthetase is then altered so that it charges the tRNACUA only with a desired unnatural amino acid. One such Orthogonal Pair for use in E. coli was developed from the tRNA2/GlnRS Pair from Saccharomyces cereVisiae.3 The development of additional Orthogonal tRNA/aaRS Pairs may allow the simultaneous incorporation of multiple unnatural amino acids into proteins. Moreover, different aminoacyl synthetases may be better starting points for generating active sites with particular specificities (e.g., specificity for large hydrophobic vs small hydrophilic amino acids). To this end, we have analyzed biochemical data available for tRNATyr/TyrRS Pairs from a variety of organisms. This analysis, together with in vivo complementation assays, has afforded a new Orthogonal tRNACUA Tyr /TyrRS Pair as well as insights into the development of additional Pairs. The identity elements of prokaryotic tRNATyr include a long variable arm in contrast to the short arm of eukaryotic tRNATyr.4 In addition, eukaryotic tRNATyr contains a C1:G72 positive recognition element, whereas prokaryotic tRNATyr has no such consensus base Pair.5,6 In vitro studies have also shown that tRNATyr of S. cereVisiae7 and Homo sapiens8 cannot be aminoacylated by bacterial synthetases, nor do their TyrRS aminoacylate bacterial tRNA. To test whether tRNACUA Tyr /TyrRS Pairs from these organisms are Orthogonal in E. coli, an in vivo complementation assay was used that is based on suppression of an amber stop codon in a nonessential position of the TEM-1 â-lactmase gene encoded in plasmid pBLAM.3 If the newly introduced suppressor tRNACUA is aminoacylated by any endogenous E. coli synthetases, cells will grow in the presence of ampicillin. After expressing these tRNACUA Tyr in E. coli strain DH10B transformed with pBLAM, cells survive at very high concentrations of ampicillin, greater than 1206 μg/mL (interpolated from IC50 curves in Figure 1) for tRNACUA Tyr derived from S. cereVisiae and 234 μg/mL for that from H. sapiens. When S. cereVisiae tRNACUA Gln , which is an Orthogonal tRNA, is tested under the same conditions, the cells survive at only 20 μg/mL ampicillin.3 For comparison, E. coli strains bearing pBLAM alone survive up to 9.7 μg/mL ampicillin (in the absence of any suppressor tRNA). Since the recognition of tRNA by synthetase depends on relative concentrations in the cell,9,10 the concentration of tRNACUA Tyr was decreased by expressing its gene under the weaker lac promoter instead of the strong lpp promoter. The IC50’s decreased to 383 and 84 μg/ mL ampicillin for S. cereVisiae and H. sapiens, respectively, but these values are still potentially too high to allow the use of these tRNAs in Orthogonal Pairs. The change of one single nucleotide in the anticodon (G34 to C34) made the S. cereVisiae and H. sapiens tRNATyr susceptible to acylation by the E. coli synthetases. Most tRNAs have positive and negative elements in the acceptor and anticodon domains to ensure accurate aminoacylation.11,12 Once the anticodon is changed from GUA to CUA, it is possible that noncognate synthetases that recognize tRNAs with similar anticodons have a stronger affinity for tRNACUA Tyr . We hypothesized that the introduction of * To whom correspondence should be addressed. Telephone: (858) 7849300. Fax: (858) 784-9440. E-mail: schultz@scripps.edu. † Present address: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138. (1) Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem., Int. Ed. Engl. 1995, 34, 621. (2) Liu, D. R.; Magliery, T. J.; Pastrnak, M.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10092. (3) Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780. (4) Himeno, H.; Hasegawa, T.; Ueda, T.; Watanabe, K.; Shimizu, M. Nucleic Acids Res. 1990, 18, 6815. (5) Lee, C. P.; RajBhandary, U. L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11378. (6) Quinn, C. L.; Tao, N.; Schimmel, P. Biochemistry 1995, 34, 12489. (7) Wakasugi, K.; Quinn, C. L.; Tao, N.; Schimmel, P. EMBO J. 1998, 17, 297. (8) Kleeman, T. A.; Wei, D.; Simpson, K. L.; First, E. A. J. Biol. Chem. 1997, 272, 14420. (9) Swanson, R.; Hoben, P.; Sumner-Smith, M.; Uemura, H.; Watson, L.; Soll, D. Science 1988, 242, 1548. (10) Sherman, J. M.; Rogers, M. J.; Soll, D. Nucleic Acids Res. 1992, 20, 1547. (11) Schimmel, P.; Giege, R.; Moras, D.; Yokoyama, S. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8763. (12) Giege, R.; Sissler, M.; Florentz, C. Nucleic Acids Res. 1998, 26, 5017. Figure 1. In vivo complementation assays to determine the Orthogonality of tRNACUA Tyr ’s from various organisms expressed in E. coli. pACXYCUA was used to express tRNACUA Tyr of organism X under the control of the lpp promoter where X ) Y is S. cereVisiae, X ) H is H. sapiens and X ) J is M. jannaschii. pAC-lacXYCUA is similar to pAC-XYCUA except the lpp promoter was changed to lac. pAC-supF was used to express E. coli supF tRNA. pAC is a control plasmid without any tRNA gene. pBLAM was used to express the â-lactamase gene with an amber stop codon at Ala-184. pBLAM-XYRS encodes the indicated TyrRS gene. Numbers following legend text are IC50 values in units of μg/mL ampicillin. 5010 J. Am. Chem. Soc. 2000, 122, 5010-5011
John D Fisk - One of the best experts on this subject based on the ideXlab platform.
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directed evolution of the methanosarcina barkeri pyrrolysyl trna aminoacyl trna synthetase Pair for rapid evaluation of sense codon reassignment potential
International Journal of Molecular Sciences, 2021Co-Authors: David G Schwark, Margaret A Schmitt, John D FiskAbstract:Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to sense codons in Escherichia coli. Predicting which sense codons are most amenable to reassignment and which Orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the Methanosarcina barkeri pyrrolysyl Orthogonal tRNA/aaRS Pair that activates and incorporates tyrosine. The evolved M. barkeri tRNA/aaRS Pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in E. coli DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating Methanocaldococcus jannaschii Orthogonal Pair. The new Orthogonal Pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved M. barkeri machinery are compared with related measurements employing the M. jannaschii Orthogonal Pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the M. jannaschii tyrosyl and M. barkeri pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different Orthogonal Pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the M. barkeri system will highlight the most promising positions at which the M. barkeri Orthogonal Pair may infiltrate the E. coli genetic code.
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dissecting the contribution of release factor interactions to amber stop codon reassignment efficiencies of the methanocaldococcus jannaschii Orthogonal Pair
Genes, 2018Co-Authors: David G Schwark, Margaret A Schmitt, John D FiskAbstract:Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii Orthogonal Pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.
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mapping the plasticity of the escherichia coli genetic code with Orthogonal Pair directed sense codon reassignment
Biochemistry, 2018Co-Authors: Margaret A Schmitt, Wil Biddle, John D FiskAbstract:The relative quantitative importance of the factors that determine the fidelity of translation is largely unknown, which makes predicting the extent to which the degeneracy of the genetic code can be broken challenging. Our strategy of using Orthogonal tRNA/aminoacyl tRNA synthetase Pairs to precisely direct the incorporation of a single amino acid in response to individual sense and nonsense codons provides a suite of related data with which to examine the plasticity of the code. Each directed sense codon reassignment measurement is an in vivo competition experiment between the introduced Orthogonal translation machinery and the natural machinery in Escherichia coli. This report discusses 20 new, related genetic codes, in which a targeted E. coli wobble codon is reassigned to tyrosine utilizing the Orthogonal tyrosine tRNA/aminoacyl tRNA synthetase Pair from Methanocaldococcus jannaschii. One at a time, reassignment of each targeted sense codon to tyrosine is quantified in cells by measuring the fluoresc...
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Mapping the Plasticity of the Escherichia coli Genetic Code with Orthogonal Pair-Directed Sense Codon Reassignment
2018Co-Authors: Margaret A. Schmitt, Wil Biddle, John D FiskAbstract:The relative quantitative importance of the factors that determine the fidelity of translation is largely unknown, which makes predicting the extent to which the degeneracy of the genetic code can be broken challenging. Our strategy of using Orthogonal tRNA/aminoacyl tRNA synthetase Pairs to precisely direct the incorporation of a single amino acid in response to individual sense and nonsense codons provides a suite of related data with which to examine the plasticity of the code. Each directed sense codon reassignment measurement is an in vivo competition experiment between the introduced Orthogonal translation machinery and the natural machinery in Escherichia coli. This report discusses 20 new, related genetic codes, in which a targeted E. coli wobble codon is reassigned to tyrosine utilizing the Orthogonal tyrosine tRNA/aminoacyl tRNA synthetase Pair from Methanocaldococcus jannaschii. One at a time, reassignment of each targeted sense codon to tyrosine is quantified in cells by measuring the fluorescence of GFP variants in which the essential tyrosine residue is encoded by a non-tyrosine codon. Significantly, every wobble codon analyzed may be partially reassigned with efficiencies ranging from 0.8 to 41%. The accumulation of the suite of data enables a qualitative dissection of the relative importance of the factors affecting the fidelity of translation. While some correlation was observed between sense codon reassignment and either competing endogenous tRNA abundance or changes in aminoacylation efficiency of the altered Orthogonal system, no single factor appears to predominately drive translational fidelity. Evaluation of relative cellular fitness in each of the 20 quantitatively characterized proteome-wide tyrosine substitution systems suggests that at a systems level, E. coli is robust to missense mutations
Shaohui Foong - One of the best experts on this subject based on the ideXlab platform.
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lateral optical sensor with slip detection for locating live products on moving conveyor
IEEE Transactions on Automation Science and Engineering, 2010Co-Authors: Kokmeng Lee, Shaohui FoongAbstract:This paper presents a method to determine the 2-D profile and motion of a live product (such as chicken for poultry meat processing) on a moving conveyor from a lateral optical sensor that consists of an Orthogonal Pair of line array (LA) scanners. Unlike most line array (LA) scanners designed to provide a 2-D image of a static object, the lateral optical sensor presented here offers a practical means to detect object slippage on the conveyor in real time. Three examples are given to illustrate the effectiveness of this sensing method. The first simulates the 2-D boundary of a geometrically well-defined object on an accelerating conveyor, which offers intuitive insights on the effects of conveyor dynamics and object slippage on the accuracy of the 2-D boundary measurement. The second experimentally demonstrates the extendibility of LA sensors to detect both engineering and natural objects. The final example illustrates the application of the lateral optical sensor as a real time feedback sensor for active singulation of natural objects.
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lateral optical sensor with slip detection of natural objects on moving conveyor
International Conference on Robotics and Automation, 2008Co-Authors: Kokmeng Lee, Shaohui FoongAbstract:This paper presents a method to determine the 2D profile and velocity of an object on a moving conveyor from a lateral optical sensor that consists of an Orthogonal Pair of line array (LA) scanners. Unlike most LA scanners which are designed to provide a 2D image of a static object, the lateral optical sensor presented here offers an additional and practical means to detect object slippage on the conveyor in real time. We illustrate numerically the effectiveness of this sensing method with two illustrative examples. The first simulates the 2D boundary of a geometrically well-defined object on an accelerating conveyor, which offers intuitive insights on the effects of conveyor dynamics and object slippage on the accuracy of the 2D boundary measurement. The second demonstrates the application of the lateral optical sensor as a real time feedback sensor for active singulation of natural objects.
Margaret A Schmitt - One of the best experts on this subject based on the ideXlab platform.
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directed evolution of the methanosarcina barkeri pyrrolysyl trna aminoacyl trna synthetase Pair for rapid evaluation of sense codon reassignment potential
International Journal of Molecular Sciences, 2021Co-Authors: David G Schwark, Margaret A Schmitt, John D FiskAbstract:Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to sense codons in Escherichia coli. Predicting which sense codons are most amenable to reassignment and which Orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the Methanosarcina barkeri pyrrolysyl Orthogonal tRNA/aaRS Pair that activates and incorporates tyrosine. The evolved M. barkeri tRNA/aaRS Pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in E. coli DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating Methanocaldococcus jannaschii Orthogonal Pair. The new Orthogonal Pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved M. barkeri machinery are compared with related measurements employing the M. jannaschii Orthogonal Pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the M. jannaschii tyrosyl and M. barkeri pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different Orthogonal Pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the M. barkeri system will highlight the most promising positions at which the M. barkeri Orthogonal Pair may infiltrate the E. coli genetic code.
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dissecting the contribution of release factor interactions to amber stop codon reassignment efficiencies of the methanocaldococcus jannaschii Orthogonal Pair
Genes, 2018Co-Authors: David G Schwark, Margaret A Schmitt, John D FiskAbstract:Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii Orthogonal Pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.
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mapping the plasticity of the escherichia coli genetic code with Orthogonal Pair directed sense codon reassignment
Biochemistry, 2018Co-Authors: Margaret A Schmitt, Wil Biddle, John D FiskAbstract:The relative quantitative importance of the factors that determine the fidelity of translation is largely unknown, which makes predicting the extent to which the degeneracy of the genetic code can be broken challenging. Our strategy of using Orthogonal tRNA/aminoacyl tRNA synthetase Pairs to precisely direct the incorporation of a single amino acid in response to individual sense and nonsense codons provides a suite of related data with which to examine the plasticity of the code. Each directed sense codon reassignment measurement is an in vivo competition experiment between the introduced Orthogonal translation machinery and the natural machinery in Escherichia coli. This report discusses 20 new, related genetic codes, in which a targeted E. coli wobble codon is reassigned to tyrosine utilizing the Orthogonal tyrosine tRNA/aminoacyl tRNA synthetase Pair from Methanocaldococcus jannaschii. One at a time, reassignment of each targeted sense codon to tyrosine is quantified in cells by measuring the fluoresc...
Osamu Nureki - One of the best experts on this subject based on the ideXlab platform.
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pyrrolysyl trna synthetase trna pyl structure reveals the molecular basis of Orthogonality
Nature, 2009Co-Authors: Kayo Nozawa, Patrick Odonoghue, Sarath Gundllapalli, Yuhei Araiso, Ryuichiro Ishitani, Takuya Umehara, Dieter Soll, Osamu NurekiAbstract:Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNA(Pyl) (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-tRNA(Pyl) (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code. This technology relies on the directed evolution of an 'Orthogonal' tRNA synthetase-tRNA Pair in which an engineered aminoacyl-tRNA synthetase (aaRS) specifically and exclusively acylates the Orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and tRNA(Pyl) function as an Orthogonal Pair in vivo. Here we show that Desulfitobacterium hafniense PylRS-tRNA(Pyl) is an Orthogonal Pair in vitro and in vivo, and present the crystal structure of this Orthogonal Pair. The ancient emergence of PylRS-tRNA(Pyl) allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS-tRNA interaction surface that is highly distinct from those observed in any other known aaRS-tRNA complex; it is this general property that underlies the molecular basis of Orthogonality.