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Languages : en
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Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are: (1) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs), and (2) an aminoacyl-tRNA synthetase, which aminoacylates the suppressor tRNA but no other tRNA in the cell. Here, we describe two such aaRS/suppressor tRNA pairs, one for use in the yeast S. cerevisiae, and another for use in E. coli. The '21st synthetase/tRNA pairs' include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast.
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Languages : en
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Book Description
Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are: (1) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs), and (2) an aminoacyl-tRNA synthetase, which aminoacylates the suppressor tRNA but no other tRNA in the cell. Here, we describe two such aaRS/suppressor tRNA pairs, one for use in the yeast S. cerevisiae, and another for use in E. coli. The '21st synthetase/tRNA pairs' include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast.
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Languages : en
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Our objective is to develop general methods for the site-specific incorporation of amino acid analogues into proteins in bacterial and in eukaryotic cells. The approach consists of the use of an amber suppressor transfer RNA (tRNA) aminoacylated with an amino acid analogue, with the help of a mutant aminoacyl-tRNA synthetase, to insert the amino acid analogue at a specific site in a protein. The site of insertion of the analogue is specified by an appropriately placed amber termination codon within the gene for the protein of interest. This approach has two key requirements: (1) an amber suppressor tRNA, which can not be aminoacylated by any of the endogenous aminoacyltRNA synthetases and (2) an aminoacyi-tRNA synthetase, which aminoacylates the amber suppressor tRNA but no other tRNA in the cell. Therefore, an important first goal is to identify such a 21st aminoacyl-tRNA synthetase tRNA synthetase-amber tRNA pair. This goal has been achieved. Several mutations have been introduced into yeast tyrosyl-tRNA synthetase (TyrRS) for isolating mutants that incorporate iodotyrosine into tRNA instead of tyrosine. Work on an alternative approach applicable to mammalian cells has led to a possible general approach for the introduction of two different amino acid analogues into a protein.
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Languages : en
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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.
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Publisher: Academic Press
ISBN: 0080921639
Category : Science
Languages : en
Pages : 334
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Book Description
By combining the tools of organic chemistry with those of physical biochemistry and cell biology, Non-Natural Amino Acids aims to provide fundamental insights into how proteins work within the context of complex biological systems of biomedical interest. The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. With more than 400 volumes published, each Methods in Enzymology volume presents material that is relevant in today's labs -- truly an essential publication for researchers in all fields of life sciences. Demonstrates how the tools and principles of chemistry combined with the molecules and processes of living cells can be combined to create molecules with new properties and functions found neither in nature nor in the test tube Presents new insights into the molecular mechanisms of complex biological and chemical systems that can be gained by studying the structure and function of non-natural molecules Provides a "one-stop shop" for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods
Author: Kiket Avi Omer Makoubi
Publisher:
ISBN:
Category :
Languages : en
Pages : 506
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Book Description
Author: Wallace M. Pasika
Publisher:
ISBN: 9780120252022
Category : Science
Languages : en
Pages : 278
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Book Description
Author: Amrita Singh
Publisher:
ISBN:
Category :
Languages : en
Pages : 430
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Book Description
The field of protein engineering has been greatly augmented by the expansion of the genetic code using unnatural amino acids as well as the development of cell-free synthesis systems with high protein yield. Cell-free synthesis systems have improved considerably since they were first described almost 40 years ago. Residue specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an amino acid depleted cell-free protein synthesis system that can be used to study residue specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines high protein expression yields with a high level of analog substitution in the target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid-depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo format. We use this amino acid depleted cell-free synthesis system for the directed evolution of streptavidin, a protein that finds wide application in molecular biology and biotechnology. We evolve streptavidin using in vitro compartmentalization in emulsions to bind to desthiobiotin and find, at the conclusion of our experiment, that our evolved streptavidin variants are capable of binding to both biotin and desthiobiotin equally well. We also discover a set of mutations for streptavidin that are potentially powerful stabilizing mutations that we believe will be of great use to the greater research community.
Author: Nediljko Budisa
Publisher: John Wiley & Sons
ISBN: 3527607099
Category : Science
Languages : en
Pages : 312
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Book Description
The ability to introduce non-canonical amino acids in vivo has greatly expanded the repertoire of accessible proteins for basic research and biotechnological application. Here, the different methods and strategies to incorporate new or modified amino acids are explained in detail, including a lot of practical advice for first-time users of this powerful technique. Novel applications in protein biochemistry, genomics, biotechnology and biomedicine made possible by the expansion of the genetic code are discussed and numerous examples are given. Essential reading for all molecular life scientists who want to stay ahead in their research.
Author: Arnaud Gautier
Publisher: Humana Press
ISBN: 9781493922710
Category : Science
Languages : en
Pages : 0
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Book Description
This detailed volume provides in-depth protocols for protein labeling techniques and applications, with an additional focus on general background information on the design and generation of the organic molecules used for the labeling step. Chapters provide protocols for labeling techniques and applications, with an additional focus on general background information on the design and generation of the organic molecules used for the labeling step. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Site-Specific Protein Labeling: Methods and Protocols provides a comprehensive overview on the most relevant and established labeling methodologies, and helps researchers to choose the most appropriate labeling method for their biological question.
Author: James Samuelson
Publisher: Humana Press
ISBN: 9781493962877
Category : Science
Languages : en
Pages : 266
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Book Description
This book provides guidance to those wishing to create enzyme variants. It covers such topics as a simple method for generating site-specific mutations within bacterial chromosomes and the engineering of two difference types of rare-cutting endonucleases.
