Showing papers by "Peter G. Schultz published in 2007"

Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines

Abstract: The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK

Abstract: Constitutive overexpression and activation of NPM-ALK fusion protein [t(2:5)(p23;q35)] is a key oncogenic event that drives the survival and proliferation of anaplastic large-cell lymphomas (ALCLs). We have identified a highly potent and selective small-molecule ALK inhibitor, NVP-TAE684, which blocked the growth of ALCL-derived and ALK-dependent cell lines with IC50 values between 2 and 10 nM. NVP-TAE684 treatment resulted in a rapid and sustained inhibition of phosphorylation of NPM-ALK and its downstream effectors and subsequent induction of apoptosis and cell cycle arrest. In vivo, NVP-TAE684 suppressed lymphomagenesis in two independent models of ALK-positive ALCL and induced regression of established Karpas-299 lymphomas. NVP-TAE684 also induced down-regulation of CD30 expression, suggesting that CD30 may be used as a biomarker of therapeutic NPM-ALK kinase activity inhibition.

Genetic incorporation of unnatural amino acids into proteins in mammalian cells

Abstract: The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate unnatural amino acids into proteins in mammalian host cells, for example, primate host cells and rodent host cells. The invention provides, for example but not limited to, translation systems that include host cells (e.g., primate or rodent cells), orthogonal aminoacyl-tRNA synthetases derived from eubacterial synthetases, orthogonal tRNAs, and the unnatural amino acid. The invention also relates to methods for producing proteins of interest comprising at least one unnatural amino acid in mammalian host cell systems.

Essential role of stromally induced hedgehog signaling in B-cell malignancies

Abstract: Interaction of cancer cells with their microenvironment generated by stromal cells is essential for tumor cell survival and influences the localization of tumor growth. Here we demonstrate that hedgehog ligands secreted by bone-marrow, nodal and splenic stromal cells function as survival factors for malignant lymphoma and plasmacytoma cells derived from transgenic Emu-Myc mice or isolated from humans with these malignancies. Hedgehog pathway inhibition in lymphomas induced apoptosis through downregulation of Bcl2, but was independent of p53 or Bmi1 expression. Blockage of hedgehog signaling in vivo inhibited expansion of mouse lymphoma cells in a syngeneic mouse model and reduced tumor mass in mice with fully developed disease. Our data indicate that stromally induced hedgehog signaling may provide an important survival signal for B- and plasma-cell malignancies in vitro and in vivo. Disruption of this interaction by hedgehog pathway inhibition could provide a new strategy in lymphoma and multiple myeloma therapy.

Control of protein phosphorylation with a genetically encoded photocaged amino acid

Abstract: We genetically encoded the photocaged amino acid 4,5-dimethoxy-2-nitrobenzylserine (DMNB-Ser) in Saccharomyces cerevisiae in response to the amber nonsense codon TAG. This amino acid was converted to serine in living cells by irradiation with relatively low-energy blue light and was used to noninvasively photoactivate phosphorylation of the transcription factor Pho4, which controls the cellular response to inorganic phosphate. When substituted at phosphoserine sites that control nuclear export of Pho4, blocks phosphorylation and subsequent export by the receptor Msn5 (ref. 2). We triggered phosphorylation of individual serine residues with a visible laser pulse and monitored nuclear export of Pho4-GFP fusion constructs in real time. We observed distinct export kinetics for differentially phosphorylated Pho4 mutants, which demonstrates dynamic regulation of Pho4 function. This methodology should also facilitate the analysis of other cellular processes involving free serine residues, including catalysis, biomolecular recognition and ion transport.

A genomic screen for activators of the antioxidant response element.

Abstract: The antioxidant response element (ARE) is a cis-acting regulatory enhancer element found in the 5′ flanking region of many phase II detoxification enzymes. Up-regulation of ARE-dependent target genes is known to have neuroprotective effects; yet, the mechanism of activation is largely unknown. By screening an arrayed collection of ≈15,000 full-length expression cDNAs in the human neuroblastoma cell line IMR-32 with an ARE-luciferase reporter, we have identified several cDNAs not previously associated with ARE activation. A subset of cDNAs, encoding sequestosome 1 (SQSTM1) and dipeptidylpeptidase 3 (DPP3), activated the ARE in primary mouse-derived cortical neurons. Overexpression of SQSTM1 and DPP3 in IMR-32 cells stimulated NF-E2-related factor 2 (NRF2) nuclear translocation and led to increased levels of NAD(P)H:quinone oxidoreductase 1, a protein which is transcriptionally regulated by the ARE. When transfected into IMR-32 neuroblastoma cells that were depleted of transcription factor NRF2 by RNA interference, SQSTM1 and DPP3 were unable to activate the ARE or induce NAD(P)H:quinone oxidoreductase 1 expression, indicating that the ARE activation upon ectopic expression of these cDNAs is mediated by NRF2. Studies with pharmacological inhibitors indicated that 1-phosphatidylinositol 3-kinase and protein kinase C signaling are essential for activity. Overexpression of these cDNAs conferred partial resistance to hydrogen peroxide or rotenone-induced toxicity, consistent with the induction of antioxidant and phase II detoxification enzymes, which can protect from oxidative stress. This work and other such studies may provide mechanisms for activating the ARE in the absence of general oxidative stress and a yet-unexploited therapeutic approach to degenerative diseases and aging.

Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

Abstract: This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Small-molecule synergist of the Wnt/β-catenin signaling pathway

Abstract: The Wnt/β-catenin signaling pathway regulates cell fate and behavior during embryogenesis, adult tissue homeostasis, and regeneration. When inappropriately activated, the pathway has been linked to colorectal cancer and melanoma, and when attenuated it may contribute to Alzheimer's disease and osteoporosis. Small molecules that modulate Wnt signaling will likely provide new insights into the regulation of this key developmental pathway and ultimately provide pharmacological agents to control Wnt signaling in vivo. To this end, we screened a library of 100,000 small molecules for activity in a cell-based assay of Wnt/β-catenin signaling and discovered a purine derivative, QS11, that synergizes with Wnt-3a ligand in the activation of Wnt/β-catenin signal transduction. Through affinity chromatography and subsequent functional assays, we showed that QS11 binds and inhibits the GTPase activating protein of ADP-ribosylation factor 1 (ARFGAP1), suggesting that QS11 modulates Wnt/β-catenin signaling through an effect on protein trafficking. Consistent with its function as an ARFGAP inhibitor, QS11 inhibits migration of ARFGAP overexpressing breast cancer cells.

Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y730 and Y731 in Radical Propagation

Abstract: E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: α2 and β2. α2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. β2 contains the stable diiron tyrosyl radical (Y122·) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122· in β2 and C439 in the active site of α2. A docking model of α2β2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in α2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-α2 and Y731NH2Y-α2 have been generated with...

Inhibition of histone deacetylase activity induces developmental plasticity in oligodendrocyte precursor cells

Abstract: Recently, it was demonstrated that lineage-committed oligodendrocyte precursor cells (OPCs) can be converted to multipotent neural stem-like cells, capable of generating both neurons and glia after exposure to bone morphogenetic proteins. In an effort to understand and control the developmental plasticity of OPCs, we developed a high-throughput screen to identify novel chemical inducers of OPC reprogramming. Using this system, we discovered that inhibition of histone deacetylase (HDAC) activity in OPCs acts as a priming event in the induction of developmental plasticity, thereby expanding the differentiation potential to include the neuronal lineage. This conversion was found to be mediated, in part, through reactivation of sox2 and was highly reproducible at the clonal level. Further, genome-wide expression analysis demonstrated that HDAC inhibitor treatment activated sox2 and 12 other genes that identify or maintain the neural stem cell state while simultaneously silencing a large group of oligodendrocyte lineage-specific genes. This series of experiments demonstrates that global histone acetylation, induced by HDAC inhibition, can partially reverse the lineage restriction of OPCs, thereby inducing developmental plasticity.

Expanding the eukaryotic genetic code

Abstract: This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Reversine increases the plasticity of lineage-committed mammalian cells.

Abstract: Previously, a small molecule, reversine, was identified that reverses lineage-committed murine myoblasts to a more primitive multipotent state. Here, we show that reversine can increase the plasticity of C2C12 myoblasts at the single-cell level and that reversine-treated cells gain the ability to differentiate into osteoblasts and adipocytes under lineage-specific inducing conditions. Moreover, reversine is active in multiple cell types, including 3T3E1 osteoblasts and human primary skeletal myoblasts. Biochemical and cellular experiments suggest that reversine functions as a dual inhibitor of nonmuscle myosin II heavy chain and MEK1, and that both activities are required for reversine's effect. Inhibition of MEK1 and nonmuscle myosin II heavy chain results in altered cell cycle and changes in histone acetylation status, but other factors also may contribute to the activity of reversine, including activation of the PI3K signaling pathway.

A genetically encoded metabolically stable analogue of phosphotyrosine in Escherichia coli.

Abstract: p-Carboxymethyl- l-phenylalanine ( pCMF), a phosphotyrosine (pTyr) mimetic that is resistant to protein tyrosine phosphatase hydrolysis, was cotranslationally incorporated into proteins in Escherichia coli using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. The pCMF-specific aaRS was identified from a large library of Methanococcus jannaschii tyrosyl-tRNA synthetase active-site mutants by a combination of positive and negative genetic selections. When pCMF was substituted for Tyr701 in human signal transducer and activator of transcription-1 (STAT1), a constitutively active mutant was obtained that dimerizes and binds a DNA oligonucleotide duplex that contains the M67 site recognized by Tyr701-phosphorylated STAT1. Genetic incorporation of pCMF into proteins should provide a new tool for the preparation of stable analogues of a wide array of phosphoproteins involved in signal transduction pathways, as well as the development of peptide-based, cellularly expressed inhibitors of...

A genetically encoded diazirine photocrosslinker in Escherichia coli.

Abstract: Photoaffinity labels and crosslinkers have been used to map biomolecular interactions, as well as to identify the biological targets of small molecules. Benzophenones are among the most useful photocrosslinking agents and preferentially insert into C H bonds upon excitation with UV light. Aryl azides and [3-(trifluoromethyl)-3H-diazin-3yl]phenones generate reactive nitrenes and carbenes, respectively, that undergo relatively nonspecific insertion and addition reactions. 6] To facilitate the selective incorporation of photocrosslinking agents into proteins in living cells, we recently genetically encoded para-benzoyl-l-phenylalanine (pBpa) and para-azido-l-phenylalanine (pAzpa) in response to the amber nonsense codon in E. coli, yeast and mammalian cells. These photocrosslinkers were site-specifically incorporated into proteins by means of heterologous amber suppressor tRNA/aminoacyl–tRNA synthetase (aaRS) pairs that recognize the unnatural amino acid, but do not cross-react with endogenous host cell tRNAs, aaRSs or amino acids. To expand the photoaffinity label repertoire, we now report that 4’-[3-(trifluoromethyl)-3H-diazirin-3-yl]-l-phenylalanine (TfmdPhe; Figure 1A) can also be genetically encoded with excellent efficiency and fidelity in bacteria. TfmdPhe has useful photochemical properties and is stable under physiological conditions. Upon excitation by ~350 nm light, TfmdPhe undergoes fragmentation to N2 and a reactive carbene, which readily inserts into C H or O H bonds. In contrast, pAzpa requires relatively short wavelength UV excitation and can rearrange to less reactive secondary products prior to crosslinking. TfmdPhe is also somewhat smaller than pBpa, which can facilitate its incorporation into proteins at sites that are involved in biomolecular interactions. TfmdPhe has been incorporated into proteins previously by a chemically misacylated amber suppressor tRNA in a cell-free protein expression system. However, this method severely limits the protein yield, and is not amenable to studies directly in living cells. TfmdPhe was synthesized by using a previously reported method. To selectively incorporate TfmdPhe into proteins in E. coli, an orthogonal amber suppressor tRNA/aaRS was generated from a Methanococcus jannaschii amber suppressor tRNA (MjtRNACUA)/para-bromo-l-phenylalanyl-tRNA synthetase (BrPheRS) pair by using a previously reported, directed evolution strategy. BrPheRS was used as a template for mutagenesis because para-bromo-l-phenylalanine (BrPhe) is structurally similar to TfmdPhe, and BrPheRS has polypeptide backbone ACHTUNGTRENNUNGrearrangements that enlarge the substrate-binding pocket. Based on the structure of the BrPheRS–BrPhe complex, a library of aaRS active-site mutants was generated, in which residues L32, L65, H70, Q109, H160 and Y161 of BrPheRS were randomized by overlap extension polymerase chain reaction with synthetic oligonucleotide primers; the intended mutations were encoded by NNK (N=A+T+C+G; K=T+G). This library of aaRS mutants (on pBK plasmids under the control of the E. coli GlnRS promoter and terminator) was then passed through rounds of alternating positive and negative selections. In the positive selection, cell survival is dependent on the suppression of an amber codon that was introduced at a permissive site in the chloramphenicol acetyl transferase (CAT) gene in the presence of chloramphenicol and 1 mm TfmdPhe. The negative selection utilizes the toxic barnase gene with amber mutations at two permissive sites, and is carried out in the absence of TfmdPhe. Cells that contain MjTyrRS variants that acylate MjtRNACUA with TfmdPhe, but not any endogenous amino acids survive both positive and negative selections, whereas cells that contain MjTyrRS variants that acylate MjtRNACUA with endogenous amino acids express barnase and die in the negative selection. Five rounds of selection afforded five colonies that survived on chloramphenicol only in the presence of TfmdPhe. DNA sequencing of MjTyrRS mutants in these five colonies revealed one unique TfmdPheRS that had three new mutation sites : Y32I, H70F, and Q109M as well as four mutations (E107S, D158P, I159L, and L162E) that were inherited from BrPheRS. To determine the efficiency and fidelity of the incorporation of TfmdPhe into proteins, an amber codon was substituted for [a] Dr. E. M. Tippmann , Dr. W. Liu , Dr. D. Summerer, A. V. Mack, Prof. Dr. P. G. Schultz Department of Chemistry and the Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 N. Torrey Pines Rd, La Jolla, CA 92037 (USA) Fax: (+1)858-784-9440 E-mail : schultz@scripps.edu [b] Dr. E. M. Tippmann + Present address: Cardiff School of Chemistry, Cardiff University Cardiff, Wales, CF10 3XQ (UK) [c] Dr. W. Liu + Present address: Department of Chemistry, Texas A&M University College Station, TX 77842 (USA) [] These authors contributed equally to this work. Figure 1. A) Structure of 4’-[3-(trifluoromethyl)-3H-diazin-3-yl]-l-phenylalanine (TfmdPhe); B) Specificity and efficiency of TfmdPhe incorporation by TfmdPheRS–MjtRNA CUA pair. TfmdPhe was incorporated in response to an amber codon at position 7 in Z domain, analyzed by SDS-PAGE, and stained by gel code blue. Proteins were purified by Ni-affinity chromatography.

Efforts Toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

Abstract: Expansion of the genetic alphabet has been a long-time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semisynthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogues, such as propynylisocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogues inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogues that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluorobenzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self-pair formed between two d3FB analogues is sufficient for in vitro DNA r...

In vivo selection for metastasis promoting genes in the mouse

Abstract: Here, we report the identification of a metastasis promoting factor by a forward genetic screen in mice. A retroviral cDNA library was introduced into the nonmetastatic cancer cell line 168FARN, which was then orthotopically transplanted into mouse mammary fat pads, followed by selection for cells that metastasize to the lung. The genes encoding the disulfide isomerase ERp5 and β-catenin were found to promote breast cancer invasion and metastasis. Disulfide isomerases (thiol isomerases), which catalyze disulfide bond formation, reduction, and isomerization, have not previously been implicated in cancer cell signaling and tumor metastasis. Overexpression of ERp5 promotes both in vitro migration and invasion and in vivo metastasis of breast cancer cells. These effects were shown to involve activation of ErbB2 and phosphoinositide 3-kinase (PI3K) pathways through dimerization of ErbB2. Activation of ErbB2 and PI3K subsequently stimulates RhoA and β-catenin, which mediate the migration and invasion of tumor cells. Inhibition of ErbB2 and PI3K reverses the phenotypes induced by ERp5. Finally, ERp5 was shown to be up-regulated in human surgical samples of invasive breast cancers. These data identify a link between disulfide isomerases and tumor development, and provide a mechanism that modulates ErbB2 and PI3K signaling in the promotion of cancer progression.

A Small-Molecule Antagonist of the Hedgehog Signaling Pathway

Abstract: The Hedgehog (Hh) signaling pathway plays an important role in embryonic pattern formation and adult tissue maintenance by directing cell differentiation and proliferation. In mammals, three Hh genes, Sonic (Shh), Indian (Ihh), and Desert (Dhh), have been identified. Binding of Hh protein to the membrane receptor Patched (Ptc) releases its inhibitory effect on Smoothened (Smo). Activated Smo further transduces downstream signals to activate the Gli family of transcription factors, which promote the expression of Hh signaling target genes. Hh signaling has recently attracted considerable interest based on the discovery that aberrant activation of Shh signaling leads to the formation of various tumors, which include pancreatic cancer, medulloblastoma, basal cell carcinoma, small cell lung cancer, and prostate cancer. 5] Cell-based phenotypic assays and, more recently, pathway screens of natural products and synthetic small molecules have provided useful chemical tools for modulating and/or studying complex cellular processes, both in vitro and in vivo. Several Hh antagonists, including cyclopamine, CUR61414, and SANT1-4 have been reported. Some of these antagonists exert antiproliferative effects by binding directly to Smo. However, cancer cells with mutations downstream of Smo are resistant to these antagonists. Therefore, Hh antagonists that interrupt downstream Hh signaling could lead to antiproliferative agents with a broader spectrum of activity. Here, we report the identification and characterization of the Hh signaling antagonist, JK184, and initial studies to characterize its biological mechanism of action. To screen small molecule libraries for compounds that antagonize Hh signaling, we developed a protocol using mesenchymal progenitor (C3H10T1/2) cells derived from the mouse embryonic mesoderm. These cells were stably transfected with a reporter construct that encoded luciferase, which was driven by Gli responsive elements, along with a neomycin resistanceconferring gene. Stably transfected C3H10T1/2 cells were plated into 384-well plates, and treated with a library of approximately 20000 heterocycles (2 mm, final concentration). After treatment with compound for 36 h in the presence of recombinant peptide that corresponded to the N terminus of Shh (100 ngmL ), luciferase activity was assayed, and a number of active 2,4-disubstituted thiazole compounds were identified. One of these compounds, JK184 (Figure 1A), inhibited Gli-dependent transcriptional activity in a dose-dependent manner with an IC50 value of 30 nm. This effect was fur-

Crystal structure of a biosynthetic sulfo-hirudin complexed to thrombin.

Abstract: The leech-derived anticoagulant hirudin is post-translationally sulfated on tyrosine 63, resulting in a >10-fold increase in its affinity for thrombin. We report the structure of a biosynthetic sul...

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

Abstract: The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Identification and characterization of small-molecule inducers of epidermal keratinocyte differentiation.

Abstract: An essential function of the human epidermis is the maintenance of a protective barrier against the environment. As a consequence, keratinocytes, which make up this layer of the skin, undergo an elaborate process of self-renewal, terminal differentiation, and cell death. Misregulation of these processes can lead to several human diseases, including psoriasis and basal cell and squamous cell carcinomas. To identify novel regulators of keratinocyte differentiation, a cell-based screen of small-molecule libraries was carried out for molecules that induce terminal differentiation of normal human epidermal keratinocytes. One class of molecules was identified, the 2-(3,4,5-trimethoxyphenylamino)-pyrrolo[2,3-d]pyrimidines, which were shown to induce differentiation of epidermal progenitor cells to terminally differentiated keratinocytes. These molecules serve as useful mechanistic probes of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis and may lead to novel therapeutic approaches for the treatment of skin hyperproliferative disorders.

System for the expression of orthogonal translation components in eubacterial host cells

Abstract: The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

Genetically programmed expression of selectively sulfated proteins in eubacteria

Abstract: The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid sulfotyrosine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid sulfotyrosine and translation systems.

Genetically encoded fluorescent coumarin amino acids

Abstract: The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine and related translation systems.