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Category :
Languages : en
Pages :
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Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.
Author: Spencer Jay Anthony-Cahill
Publisher:
ISBN:
Category :
Languages : en
Pages : 676
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Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 2
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A method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the biosynthesis of unnatural polymers and proteins with novel structures and activities. Our approach consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pair that are not catalytically competent with all the endogenous Escherichia coli tRNAs an aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair in E. coli has been derived from the Saccharomyces cerevisiae tRNA (sub Asp) and aspartyl-tRNA syathetase, and the in vitro and in vivo characteristics of this pair were determined. In order to achieve a high specificity for the amino acid, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers very high specificity for the amino acid of interest, Which cannot be achieved by conventional methods.
Author: Jianming Xie
Publisher:
ISBN:
Category : Molecular evolution
Languages : en
Pages : 278
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This thesis focuses on expanding the genetic code of E. coli to include unnatural amino acids beyond the common 20. By means of an orthogonal pair of amber suppressor tRNA[Special characters omitted.] /tyrosyl-tRNA synthetase (TyrRS) imported from an archaea M. jannaschii, several novel amino acids were site-specifically incorporated into proteins in response to an amber codon in E. coli, with high fidelity and efficiency. Typically, large libraries of M. jannaschii TyrRS (Mj TyrRS) mutants were first generated by randomizing 6 residues in the amino acid binding pocket. The libraries were then subjected to alternating positive and negative selections, which may afford synthetase variants that could charge the cognate tRNA[Special characters omitted.] with the unnatural amino acid but not any of the 20 natural amino acids. In one project, a heavy-atom-containing amino acid, p -iodo-L-phenylalanine, was site-specifically incorporated into proteins. The anomalously scattering iodine atom could be used to facilitate X-ray structure determination by SAD phasing. In the second project, a phosphotyrosine analogue, p -carboxymethyl-L-phenylalanine, was cotranslationally incorporated into proteins. When this unnatural amino acid was used to replace Tyr701 in human STAT1, the resulting protein dimerized and bound the same DNA sequence as Tyr701-phosphorylated STAT1. Because this amino acid is resistant to protein tyrosine phosphatase, it should be useful in the generation of stable phosphoprotein analogues, or peptide-based inhibitors to SH2 domains. In another project, a metal-chelating amino acid, bipyridylalanine, was selectively incorporated into proteins. Genetically encoding bipyridylalanine and other metal-chelating amino acids should be useful for the generation of proteins with novel or enhanced properties, such as artificial enzymes, proteins that undergo metal-ion-assisted dimerization/trimerization or folding, proteins that contain redox-active metal-ligand complexes on their surface for use in the artificial electron transfer system, and proteins that bind fluorescent or luminescent metal ions as imaging agents, or bind radionuclides for therapeutic use. A final series of experiments involved attempts to incorporate N-methyl amino acids into proteins, which could be useful in generating protease-resistant proteins. While the N-methyl amino acid was N-demethylated by metabolic enzymes in E. coli, recent experiments suggest that such amino acids are stable in yeast, presenting a possible solution to this problem.
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 55
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The goal of this project is to develop methods for the site-specific insertion in vivo of one or more amino acid analogues into proteins in eubacteria and in eukaryotes. The amino acids to be used include those that are photoactivatable, those that are fluorescent, those that carry reactive side chains such as keto groups, heavy atoms such as iodine, those that act as spectroscopic probes, and those that mimic phosphoserine, phosphothreonine, or phosphotyrosine. Besides providing a method for the production of new biomateirals with novel chemical and biological properties, proteins carrying such amino acid analogues will have wide applications in biology. These include studies on the folding, structure, stability and function of proteins, protein-protein interactions, protein localization and protein dynamics in vivo and signal transduction.
Author: Susanne Veronika Mayer
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Christopher J. Noren
Publisher:
ISBN:
Category :
Languages : en
Pages : 7
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Author: Kiket Avi Omer Makoubi
Publisher:
ISBN:
Category :
Languages : en
Pages : 506
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Author:
Publisher: Elsevier
ISBN: 0081026919
Category : Science
Languages : en
Pages : 4266
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Book Description
Comprehensive Natural Products III, Third Edition, Seven Volume Set updates and complements the previous two editions, including recent advances in cofactor chemistry, structural diversity of natural products and secondary metabolites, enzymes and enzyme mechanisms and new bioinformatics tools. Natural products research is a dynamic discipline at the intersection of chemistry and biology concerned with isolation, identification, structure elucidation, and chemical characteristics of naturally occurring compounds such as pheromones, carbohydrates, nucleic acids and enzymes. This book reviews the accumulated efforts of chemical and biological research to understand living organisms and their distinctive effects on health and medicine and to stimulate new ideas among the established natural products community. Provides readers with an in-depth review of current natural products research and a critical insight into the future direction of the field Bridges the gap in knowledge by covering developments in the field since the second edition published in 2010 Split into 7 sections on key topics to allow students, researchers and professionals to find relevant information quickly and easily Ensures that the knowledge within is easily understood by and applicable to a large audience
Author:
Publisher: Elsevier
ISBN: 0080453821
Category : Science
Languages : en
Pages : 7944
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Book Description
This work presents a definitive interpretation of the current status of and future trends in natural products—a dynamic field at the intersection of chemistry and biology concerned with isolation, identification, structure elucidation, and chemical characteristics of naturally occurring compounds such as pheromones, carbohydrates, nucleic acids, and enzymes. With more than 1,800 color figures, Comprehensive Natural Products II features 100% new material and complements rather than replaces the original work (©1999). Reviews the accumulated efforts of chemical and biological research to understand living organisms and their distinctive effects on health and medicine Stimulates new ideas among the established natural products research community—which includes chemists, biochemists, biologists, botanists, and pharmacologists Informs and inspires students and newcomers to the field with accessible content in a range of delivery formats Includes 100% new content, with more than 6,000 figures (1/3 of these in color) and 40,000 references to the primary literature, for a thorough examination of the field Highlights new research and innovations concerning living organisms and their distinctive role in our understanding and improvement of human health, genomics, ecology/environment, and more Adds to the rich body of work that is the first edition, which will be available for the first time in a convenient online format giving researchers complete access to authoritative Natural Products content
