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Genetic Incorporation Of Noncanonical Amino Acids Into Proteins For Protein Function Investigation
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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 Exploring and Expanding the Protein Universe with Non-Canonical Amino Acids by : Gustavo Fuertes
Download or read book Exploring and Expanding the Protein Universe with Non-Canonical Amino Acids written by Gustavo Fuertes and published by Frontiers Media SA. This book was released on 2023-11-01 with total page 123 pages. Available in PDF, EPUB and Kindle. Book excerpt: The site-specific incorporation of unnatural or non-canonical amino acids (ncAAs) into proteins is a universally important tool for systems bioengineering at the interface of chemistry, biology, and biotechnology. The synergistic use of ncAA and related technologies (e.g. Xeno nucleic acids) should enable: i) New opportunities to manipulate, design and elucidate protein structure, dynamics, and function. ii) A deeper understanding of natural and evolved translational systems and their importance for artificial biology. iii) The synthesis of novel biopolymers, creating a solid basis for synthetic cells, which is also an important technology in the production of new classes of medically relevant protein-based scaffolds. Research on reprogrammed protein translation has now reached an experimental and intellectual maturity: more than 200 ncAA (i.e. more than ten times larger variety than standard amino acids) have been introduced into proteins using different routes: genetic code expansion (GCE), selective pressure incorporation (SPI), chemical mutagenesis, protein semi-synthesis, and peptide synthesis.
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 Genetically Incorporated Non-Canonical Amino Acids by : Simon J. Elsässer
Download or read book Genetically Incorporated Non-Canonical Amino Acids written by Simon J. Elsässer and published by Methods in Molecular Biology. This book was released on 2023-07-07 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis by : John Tuan Ngo
Download or read book Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis written by John Tuan Ngo and published by . This book was released on 2012 with total page 256 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. This approach, often termed "residue-specific incorporation," allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. Chapter I of this thesis describes how this strategy has been used to track cellular protein synthesis with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a "pulse" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Chapter II of this thesis describes how the selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. The work in Chapter III illustrates how the extent of temporal and spatial resolution of protein labeling can be enhanced through controlled expression of mutant synthetases. Use of characterized promoters to direct transcription of mutant synthetase genes can limit labeling to relevant cells and physiological states in settings of increased complexity. Chapter IV presents a novel strategy with which ncAAs can be uniquely incorporated at the N-terminal positions of nascent proteins while excluded from insertion at internal positions. This approach permits "site-selective" tagging of cellular proteins, and its use in tagging and visualization of cell-cycle dependent protein synthesis is described. The work described throughout this thesis was designed with the objective of providing powerful and versatile methods for the study of protein synthesis in complex multicellular systems, including live animals. Thus, Chapter V considers how these strategies might be used to dissect protein synthesis in living animals.
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 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 Genetically Incorporated Non-Canonical Amino Acids by : Yu-Hsuan Tsai
Download or read book Genetically Incorporated Non-Canonical Amino Acids written by Yu-Hsuan Tsai and published by Springer Nature. This book was released on 2023-06-05 with total page 287 pages. Available in PDF, EPUB and Kindle. Book excerpt: This detailed volume explores non-canonical amino acids (ncAAs) through their site-specific incorporation by genetic code expansion (GCE). The collection provides a broad resource of methods for implementing GCE in E. coli, mammalian cells, and animals, highlighting specific applications ranging from fluorescence labeling to photocontrol and the study of protein post-translational modification. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols serves as an ideal source of methodologies that can be adapted and extended, migrated to different model systems, and combined in new ways to help explore a wide range of biological questions and to augment industrial and pharmaceutical protein engineering.
Book Synopsis Novel Strategies for Producing Proteins with Non-proteinogenic Amino Acids by : Ryan Christopher Nangreave
Download or read book Novel Strategies for Producing Proteins with Non-proteinogenic Amino Acids written by Ryan Christopher Nangreave and published by . This book was released on 2013 with total page 129 pages. Available in PDF, EPUB and Kindle. Book excerpt: The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.
Book Synopsis Chemical and Biological Synthesis by : Nick J Westwood
Download or read book Chemical and Biological Synthesis written by Nick J Westwood and published by Royal Society of Chemistry. This book was released on 2018-08-16 with total page 317 pages. Available in PDF, EPUB and Kindle. Book excerpt: Synthetic chemistry plays a central role in many areas of chemical biology; utilising recent case studies, the goal of Chemical and Biological Synthesis is to highlight the full impact that the preparation of novel reagents can have in chemical biology. Covering the synthetic approaches that can be applied across the whole field of chemical biology, this book provides synthetic chemists with the broader context to which their work contributes and the biological questions that can be addressed through it. An ideal guide for postgraduate students and researchers in synthetic organic chemistry and chemical biology, Chemical and Biological Synthesis introduces synthetic techniques and methods to those who wish to incorporate synthesis for the first time in their biology-focused research programmes.
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.
Download or read book TRNA written by Dieter Söll and published by . This book was released on 1995 with total page 572 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Non-Canonical Amino Acids as Minimal Tags for Investigating Protein Organization and Turnover by : Ingrid-Cristiana Gebura-Vreja
Download or read book Non-Canonical Amino Acids as Minimal Tags for Investigating Protein Organization and Turnover written by Ingrid-Cristiana Gebura-Vreja and published by . This book was released on 2015 with total page 161 pages. Available in PDF, EPUB and Kindle. Book excerpt: The burgeoning field of genetic code expansion provides new tools for specifically labeling proteins for a variety of applications. Clickable non-canonical amino acids (ncAA) have been refined for almost-quantitative and highly selective reactions with complementary probes. In my thesis work I have adapted genetic code expansion for two biological questions. First, I used ncAA incorporation and click reaction to test the effect of fluorescent protein (FP) tagging on the nanoscale organization of target proteins. Second, I used these tools to generate a genetically-encoded scheme for specifi...
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 Handbook of RNA Biochemistry by : Roland K. Hartmann
Download or read book Handbook of RNA Biochemistry written by Roland K. Hartmann and published by John Wiley & Sons. This book was released on 2015-06-22 with total page 1370 pages. Available in PDF, EPUB and Kindle. Book excerpt: The second edition of a highly acclaimed handbook and ready reference. Unmatched in its breadth and quality, around 100 specialists from all over the world share their up-to-date expertise and experiences, including hundreds of protocols, complete with explanations, and hitherto unpublished troubleshooting hints. They cover all modern techniques for the handling, analysis and modification of RNAs and their complexes with proteins. Throughout, they bear the practising bench scientist in mind, providing quick and reliable access to a plethora of solutions for practical questions of RNA research, ranging from simple to highly complex. This broad scope allows the treatment of specialized methods side by side with basic biochemical techniques, making the book a real treasure trove for every researcher experimenting with RNA.
Book Synopsis Cell-Free Translation Systems by : A.S. Spirin
Download or read book Cell-Free Translation Systems written by A.S. Spirin and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 254 pages. Available in PDF, EPUB and Kindle. Book excerpt: This is a unique book that describes the most recent achievements in the methodology of protein biosynthesis under cell-free conditions. Various versions of cell-free protein-synthesizing systems and their applications to production of individual proteins on a preparative scale are reviewed. The most recent, advanced methodologies, such as continuous-exchange and continuous-flow cell-free systems and novel effecting batch-format cell-free procedures, are considered. Special attention is drawn to the possibilities of structural (NMR; X-ray) analysis of various gene expression products with the use of a new generation of cell-free systems.
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.
