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Genetic Incorporation Of Unnatural Amino Acids Into Proteins
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Book Synopsis Genetic Incorporation of Unnatural Amino Acids Into Proteins by : Isaac Nnamdi Ugwumba
Download or read book Genetic Incorporation of Unnatural Amino Acids Into Proteins written by Isaac Nnamdi Ugwumba and published by . This book was released on 2010 with total page 308 pages. Available in PDF, EPUB and Kindle. Book excerpt: The introduction of unnatural amino acids with novel properties into proteins is potentially a useful tool for understanding and manipulating their functions. The development of an in vitro method for the incorporation of a fluorescent amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco), into proteins is described here.
Book Synopsis Unnatural Amino Acids by : Loredano Pollegioni
Download or read book Unnatural Amino Acids written by Loredano Pollegioni and published by Humana Press. This book was released on 2011-10-05 with total page 409 pages. Available in PDF, EPUB and Kindle. Book excerpt: Even though they are present in nature, non-proteinogenic amino acids are usually defined as unnatural or non-natural. Beside their structural diversity, interest in these compounds is due to their occurrence in nature, their biological properties, the analytical aspects, their use as probes, and their incorporation into peptides and proteins, among other reasons. Divided into five convenient sections, Unnatural Amino Acids: Methods and Protocols deals with enzymatic methods used to produce non-natural amino acids, aspects concerning the presence of unnatural amino acids in peptides with antimicrobial properties, genetic incorporation of unnatural amino acids into proteins (yeast and mammalian cells), and detection and quantification of D-amino acids and related enzymes. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Unnatural Amino Acids: Methods and Protocols serves as an ideal guide for scientists and contributes to directing the attention of researchers to the many fields of growing scientific interest in non-natural amino acids.
Download or read book Non-Natural Amino Acids written by and published by Academic Press. This book was released on 2009-07-24 with total page 334 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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
Book Synopsis Protein Engineering by : Huimin Zhao
Download or read book Protein Engineering written by Huimin Zhao and published by John Wiley & Sons. This book was released on 2021-08-23 with total page 41 pages. Available in PDF, EPUB and Kindle. Book excerpt: A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.
Book Synopsis Unnatural Amino Acid Incorporation for Genetic Code Expansion in Mammalian Cells by : Jeffrey Kunio Takimoto
Download or read book Unnatural Amino Acid Incorporation for Genetic Code Expansion in Mammalian Cells written by Jeffrey Kunio Takimoto and published by . This book was released on 2011 with total page 150 pages. Available in PDF, EPUB and Kindle. Book excerpt: The genetic code of most organisms was evolved to encode 20 amino acids. Although the ability to encode 20 amino acids provides the basis to translate proteins necessary for life, researchers are also limited to these 20 amino acids for conventional site-directed mutagenesis. The ability to encode unnatural amino acids provides researchers the ability to circumvent the limitation imposed by the genetic code. Genetically encoding unnatural amino acids provides researchers the means to not only mimic naturally occurring posttranslational modifications but also the ability to encode amino acids with new physical or chemical properties to study biological processes. The incorporation of unnatural amino acids into proteins had been developed in Escherichia coli and also in yeast. We have developed a methodology to genetically incorporate unnatural amino acids in mammalian cells in response to an amber codon (UAG). The incorporation of unnatural amino acids is high in E. coli and yeast, but the incorporation in mammalian cells is relatively low. In addition to developing the system to incorporate unnatural amino acids in mammalian cells, we have also improved suppression efficiencies by modifying the synthetase and unnatural amino acid. To incorporate unnatural amino acids in response to an amber codon, the tRNA anticodon is mutated from a GUA to a CUA. We were able to show that engineering the anticodon-binding domain of the synthetase could enhance the recognition of the tRNA and thus increased suppression efficiencies. Furthermore, by masking the carboxyl group of the amino acid by an ester group, we were able to increase the bioavailability of an unnatural amino acid to further increase suppression efficiencies. Most evolved synthetases aminoacylate unnatural amino acids that are structurally similar to the native substrate of the wild-type synthetase. We were able to adapt Methanosarcina mazei pyrrolysine synthetase (PylRS) to charge a considerable disparate amino acid from its native substrate, O-methyl-L-tyrosine. In addition, the X-ray crystal structure was solved for the evolved PylRS complexed with O-methyl-L-tyrosine at 1.75Å. This multifaceted approach provides the basis to engineer the PylRS to incorporate a significantly diverse selection of unnatural amino acids than previously anticipated.
Book Synopsis Unnatural Amino Acids by : Loredano Pollegioni
Download or read book Unnatural Amino Acids written by Loredano Pollegioni and published by Humana Press. This book was released on 2016-08-23 with total page 409 pages. Available in PDF, EPUB and Kindle. Book excerpt: Even though they are present in nature, non-proteinogenic amino acids are usually defined as unnatural or non-natural. Beside their structural diversity, interest in these compounds is due to their occurrence in nature, their biological properties, the analytical aspects, their use as probes, and their incorporation into peptides and proteins, among other reasons. Divided into five convenient sections, Unnatural Amino Acids: Methods and Protocols deals with enzymatic methods used to produce non-natural amino acids, aspects concerning the presence of unnatural amino acids in peptides with antimicrobial properties, genetic incorporation of unnatural amino acids into proteins (yeast and mammalian cells), and detection and quantification of D-amino acids and related enzymes. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Unnatural Amino Acids: Methods and Protocols serves as an ideal guide for scientists and contributes to directing the attention of researchers to the many fields of growing scientific interest in non-natural amino acids.
Book Synopsis In Vivo Incorporation of Unnatural Amino Acids Into Proteins by :
Download or read book In Vivo Incorporation of Unnatural Amino Acids Into Proteins written by and published by . This book was released on 2002 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Engineering the Genetic Code by : Nediljko Budisa
Download or read book Engineering the Genetic Code written by Nediljko Budisa and published by John Wiley & Sons. This book was released on 2006-05-12 with total page 312 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Noncanonical Amino Acids by : Edward A. Lemke
Download or read book Noncanonical Amino Acids written by Edward A. Lemke and published by . This book was released on 2018 with total page 411 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Genetic Incorporation of Unnatural Amino Acids and Its Applications by : Hyun Soo Lee
Download or read book Genetic Incorporation of Unnatural Amino Acids and Its Applications written by Hyun Soo Lee and published by . This book was released on 2009 with total page 254 pages. Available in PDF, EPUB and Kindle. Book excerpt: To probe protein-DNA interactions, (2,2'-bipyridin-5-yl)alanine (Bpy-Ala) or p-benzoyl-L-phenylalanine ( pBpa) were incorporated into the E. coli catabolite activator protein (CAP). In the presence of Cu (II) and 3-mercaptopropionic acid, a CAP mutant containing Bpy-Ala cleaved double-stranded DNA with high sequence-specificity. The other CAP mutant containing pBpa formed a covalent complex with a DNA fragment containing the consensus operator sequence upon UV irradiation. These two experiments demonstrated that Bpy-Ala and p Bpa can be used to map the molecular details of protein-nucleic acid interactions.
Book Synopsis Genetic Incorporation of Noncanonical Amino Acids Into Proteins for Protein Function Investigation by : Ying Huang
Download or read book Genetic Incorporation of Noncanonical Amino Acids Into Proteins for Protein Function Investigation written by Ying Huang and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: With the objective to functionalize proteins for the understanding of their biological roles and developing protein-based biosensors, I have been developing methods to synthesize proteins with defined modifications and applying them to study protein functional roles and generate proteins with new properties. These methods rely on the read-through of an in-frame stop codon in mRNA by a nonsense suppressor tRNA specifically acylated with a noncanoncial amino acid (NAA) by a unique aminoacyl-tRNA synthetase and the genetic incorporation of this NAA at the stop codon site. NAAs either provide chemical handles for site-specific manipulation or mimic the posttranslational modifications, which are critical for understanding cellular regulations and signal transduction. The pyrrolysine synthetase (PylRS) has been wildly used to incorporate NAAs into proteins in E. coli. Taking advantage of PylRS, I have developed method to genetically incorporate ketone-containing N-acetyl-L-lysine analog, 2-amino-8-oxononanoic acid (KetoK), into proteins for their site-specific modifications and used it to mimic the protein lysine acetylation process. I have also modified the ribosome in order to improve the amber suppression efficiency and therefore to achieve incorporation of multiple copies of NAA into one protein. By overexpressing a truncated ribosomal protein, L11C, I have demonstrated 5-fold increase of amber suppression level in E. coli, leading to higher expression levels for proteins incorporated with NAAs. I have also demonstrated this method can be applied successfully to incorporate at least 3 NAAs into one protein in E. coli. With the success of incorporating multiple NAAs into one protein, I have further introduced two distinct NAAs into one protein simultaneously. This is done by using a wild type or evolved PylRS-pylTUUA pair and an evolved M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS)-tRNACUA pair. By suppressing both UAG and UAA stop codons in one mRNA, a protein incorporated with two NAAs is synthesized with a decent yield. There is of great interest to incorporate new NAAs into proteins, which is done by library selection. By introducing both positive and negative selective markers into one plasmid, I have developed a one-plasmid selection method. In this method, the positive and negative selections are accomplished by in a single type of cells hosting a single selection plasmid.
Book Synopsis Adding Unnatural Amino Acids to the Genetic Repertoire by : Jianming Xie
Download or read book Adding Unnatural Amino Acids to the Genetic Repertoire written by Jianming Xie and published by . This book was released on 2006 with total page 278 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Synthetic DNA and RNA Programming by : Patrick O’Donoghue
Download or read book Synthetic DNA and RNA Programming written by Patrick O’Donoghue and published by MDPI. This book was released on 2019-11-19 with total page 188 pages. Available in PDF, EPUB and Kindle. Book excerpt: Dear Colleagues, Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering and omics technologies. These technologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This Special Issue on “Synthetic RNA and DNA Programming” features original research articles and reviews, highlighting novel aspects of basic molecular biology and the molecular mechanisms of disease that were uncovered by the application and development of novel synthetic biology-driven approaches.
Book Synopsis A General Method for Site-specific Incorporation of Unnatural Amino Acids Into Proteins by : Christopher J. Noren
Download or read book A General Method for Site-specific Incorporation of Unnatural Amino Acids Into Proteins written by Christopher J. Noren and published by . This book was released on 1989 with total page 7 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Enzyme Engineering by : James Samuelson
Download or read book Enzyme Engineering written by James Samuelson and published by Humana Press. This book was released on 2016-05-01 with total page 266 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Life Sciences Industry by : Basanta Kumara Bahera
Download or read book Life Sciences Industry written by Basanta Kumara Bahera and published by Springer Nature. This book was released on 2021-08-24 with total page 221 pages. Available in PDF, EPUB and Kindle. Book excerpt: Basic principles of applied life sciences such as recombinant DNA technology is used in most life sciences industries marketing bio-formulations for designing more effective protein-based drugs, such as erythropoietin and fast-acting insulin etc. In recent times genetically engineered host cells from mammal, animal and plants are also being used in life sciences industries to manufacture biologics. This book discusses the most basic as well advanced issues on biological products for successfully managing a life sciences industry. It elucidates the life cycle of biological molecules, right from the conceptual development of different types of biopolymers, and their subsequent transfer from the conical flasks in laboratory to life sciences industries for large scale production and marketing. It focuses on sustainable longevity in the life cycle of commercial biopolymers. Cumulative facts and figures in this volume would immensely help in inspiring life sciences industry promoters to monitor value chain transfer process of biologics for better profitability. Additionally, it would serve as a perusal document for the students and researchers interested in entrepreneurial ventures or having their own start-up projects for the commercialization of biologics.
Book Synopsis Protein Evolution in the Presence of an Unnatural Amino Acid by : Amrita Singh
Download or read book Protein Evolution in the Presence of an Unnatural Amino Acid written by Amrita Singh and published by . This book was released on 2012 with total page 430 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
