Download or read book Expressed Protein Ligation written by Miquel Vila-Perelló and published by Humana. This book was released on 2020-03-07 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides a comprehensive overview of Expressed Protein Ligation (EPL), detailing methods and protocols to generate site-specifically modified proteins. Chapters include an overview of the protein semi-synthesis field, as well as related areas that have contributed to the development of EPL such as protein splicing and peptide synthesis. Following the introductory chapters, the rest of the book guides readers through protocols to perform EPL reactions, methods to synthesize peptide thioesters and to perform peptide and protein ligations, label proteins inside living cells, protocols for the semi-synthesis of phorphorylated, glycosylated and ubiquitylated proteins, synthesis and assembly of assymetrically modified nucleosomes, use of ligation auxiliaries and synthesis of cyclic proteins, as well as novel desulfurization strategies and use of selective Cys side chain protection to obtain precisely modified proteins.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 cutting-edge, Expressed Protein Ligation: Methods and Protocols will ensure successful implementation of protein semi-synthesis methods to further study the structure and function of proteins.
Download or read book Split Inteins written by Henning D. Mootz and published by . This book was released on 2017 with total page 270 pages. Available in PDF, EPUB and Kindle. Book excerpt: "This volume focuses on applications of split inteins, and the progress that has been made in the past 5 years on discovery and engineering of fast and more efficient split inteins. The first few chapters in Split Inteins: Methods and Protocols explore new techniques on how to use split inteins for affinity purification of overproduced proteins, and split-intein based technologies to prepare cyclic peptides and proteins. The next few chapters discuss semisynthetic protein trans-splicing using one synthetic intein piece, synthetic intein-extein pieces used to deliver other cargos for chemical modification both of purified proteins and of proteins in living cells, as well as isotopic labeling of proteins for NMR studies, and a discussion on how protein block copolymers can be generated by protein trans-splicing to form protein hydrogels. The last few chapters deal with intein applications in transgenic plants and conditional inteins that can be regulated in artificial ways by small molecules or light, a cassette-based approach to quickly test many intein insertion positions, and a computational approach to predict new intein split sites (the approach also works for other proteins). Written in the highly successful Methods in Molecular Biology series format, chapters include introduction 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. Cutting-edge and thorough, Split Inteins: Methods and Protocols is a valuable resource that will provide guidance toward possibilities of split intein applications, explore proven and detailed protocols adaptable to various research projects, and inspire new method developments."--OCLC.
Download or read book Homing Endonucleases and Inteins written by Marlene Belfort and published by Springer Science & Business Media. This book was released on 2008-09-22 with total page 404 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides the first and only comprehensive description and detailed summary of the genetics, structure, function, mechanisms of action, evolution and engineering of homing endonucleases and inteins. These two unique protein superfamilies, which are tied together through their frequent fusion and coevolution, have generated considerable excitement for their fundamental, structural, and functional properties, their evolution as parasitic elements, and their widespread applications as gene targeting agents and as instruments for the generation of modified proteins and novel protein combinations.
Download or read book Synthetic mRNA written by Robert E. Rhoads and published by Humana. This book was released on 2016-05-29 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.
Download or read book Site Specific Protein Labeling written by Arnaud Gautier and published by Humana Press. This book was released on 2015-01-06 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Download or read book Split Inteins as Versatile Tools in Applications of Downstream Purification and Bioconjugation written by Yamin Fan (Ph. D. in chemical engineering) and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Over the past two decades, inteins have been extensively used in a wide variety of applications in biotechnology. Split inteins are a subset of inteins, which are identified more recently and expressed in two separate segments naturally. They catalyze the splicing reaction in trans upon association of the two halves. Due to their unique features, split inteins offer improved controllability and flexibility in trans-splicing and trans-cleaving over the previous tools based on contiguous inteins. The engineered split inteins would allow the development of efficient self-cleaving affinity tags for purification applications and new methods for protein conjugation. In this work, an engineered split intein derived from Nostoc punciforme (Npu) was applied in a column-free purification strategy in combination with the aggregating tag, elastin-like polypeptide (ELP), as an initial capture step for recombinant proteins expressed in E. coli. Meanwhile, on-column purification strategy using the same engineered split intein was employed for the production of value-added biosimilar target, Granulocyte-colony stimulating factor (G-CSF). To adapt the split intein-based purification platform for the production of protein therapeutics expressed in mammalian cells, multiple leader sequences were designed and screened for optimal expression and secretion of intein-tagged precursor proteins. Moreover, the extein dependency of this engineered Npu split intein was thoroughly characterized by using in solution cleaving kinetics study and Forster Resonance Energy Transfer (FRET) based high-throughput method. The information gathered guides for fast and consistent cleavage reactions among various target proteins and provides insight to the cleavage mechanism. In this work, the trans-splicing properties of split inteins were also exploited for developing novel bioconjugation methods onto fluorescent nanodiamonds. Two split inteins, Gos-TerL and GP41.1, were used for the development of N-terminal and C-terminal oriented bioconjugation schemes, respectively. The new methods would allow rapid and spontaneous immobilization of proteins onto fluorescent nanodiamond surfaces for applications such as biomedical imaging or drug delivery.
Download or read book Molecular Gels written by Richard G. Weiss and published by Springer Science & Business Media. This book was released on 2006-06-30 with total page 969 pages. Available in PDF, EPUB and Kindle. Book excerpt: "Molecular Gels: Materials with Self-Assembled Fibrillar Networks" is a comprehensive treatise on gelators, especially low molecular-mass gelators and the properties of their gels. The structures and modes of formation of the self-assembled fibrillar networks (SAFINs) that immobilize the liquid components of the gels are discussed experimentally and theoretically. The spectroscopic, rheological, and structural features of the different classes of low molecular-mass gelators are also presented. Many examples of the application of the principal analytical techniques for investigation of molecular gels (including SANS, SAXS, WAXS, UV-vis absorption, fluorescence and CD spectroscopies, scanning electron, transmission electron and optical microscopies, and molecular modeling) are presented didactically and in-depth, as are several of the theories of the stages of aggregation of individual low molecular-mass gelator molecules leading to SAFINs. Several actual and potential applications of molecular gels in disparate fields (from silicate replication of nanostructures to art conservation) are described. Special emphasis is placed on perspectives for future developments. This book is an invaluable resource for researchers and practitioners either already researching self-assembly and soft matter or new to the area. Those who will find the book useful include chemists, engineers, spectroscopists, physicists, biologists, theoreticians, and materials scientists.
Download or read book Expanding the Uses of Split inteins Through Protein Engineering written by Stanley Wong and published by . This book was released on 2013 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Chemical and Synthetic Biology Approaches to Understand Cellular Functions Part C written by and published by Academic Press. This book was released on 2020-02-10 with total page 310 pages. Available in PDF, EPUB and Kindle. Book excerpt: Chemical and Synthetic Biology Approaches to Understand Cellular Functions - Part C, Volume 633, the latest release in the Methods in Enzymology series, continues the legacy of this premier serial. This release includes sections on Next generation probes for molecular imaging in cells, Competitive binding assay for biotin and biotin derivatives, based on avidin and biotin-4-fluorescein, Converting avidin to bind ligands other than biotin, especially steroids, Chemoenzymatic Labeling Strategy, Engineered Siderophores, Small molecules to inhibit bacterial population behavior, NMR tube bioreactor, Small molecule controlled RAS activation system, Small molecule regulated Cas9, the Design and application of synthetic receptors, and much more. - Contains the authority of authors who are leaders in their field - Provides a comprehensive source on new methods and research in enzymology
Download or read book Plant Cyclotides written by and published by Academic Press. This book was released on 2015-11-24 with total page 404 pages. Available in PDF, EPUB and Kindle. Book excerpt: Advances in Botanical Research publishes in-depth and up-to-date reviews on a wide range of topics in plant sciences. Currently in its 76th volume, the series features several reviews by recognized experts on all aspects of plant genetics, biochemistry, cell biology, molecular biology, physiology and ecology. - Publishes in-depth and up-to-date reviews on a wide range of topics in plant sciences - Contains commentary by recognized experts on all aspects of plant genetics, biochemistry, cell biology, molecular biology, physiology, and ecology - This volume features reviews of the fast moving field of plant cyclotides
Download or read book Advances in Synthetic Biology written by Vijai Singh and published by Springer Nature. This book was released on 2020-04-13 with total page 349 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book addresses the design of emerging conceptual tools, technologies and systems including novel synthetic parts, devices, circuits, oscillators, biological gates, and small regulatory RNAs (riboregulators and riboswitches), which serve as versatile control elements for regulating gene expression. Synthetic biology, a rapidly growing field that involves the application of engineering principles in biology, is now being used to develop novel systems for a wide range of applications including diagnostics, cell reprogramming, therapeutics, enzymes, vaccines, biomaterials, biofuels, fine chemicals and many more. The book subsequently summarizes recent developments in technologies for assembling synthetic genomes, minimal genomes, synthetic biology toolboxes, CRISPR-Cas systems, cell-free protein synthesis systems and microfluidics. Accordingly, it offers a valuable resource not only for beginners in synthetic biology, but also for researchers, students, scientists, clinicians, stakeholders and policymakers interested in the potential held by synthetic biology.
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
Download or read book E coli Gene Expression Protocols written by Peter E. Vaillancourt and published by Humana Press. This book was released on 2002-10-22 with total page 347 pages. Available in PDF, EPUB and Kindle. Book excerpt: Peter E. Vaillancourt presents a collection of popular and emerging methodologies that take advantage of E. coli's ability to quickly and inexpensively express recombinant proteins. The authors focus on two areas of interest: the use of E. coli vectors and strains for production of pure, functional protein, and the use of E. coli as host for the functional screening of large collections of proteins and peptides. Among the cutting-edge techniques demonstrated are those for rapid high-level expression and purification of soluble and functional recombinant protein and those essential to functional genomics, proteomics, and protein engineering.
Download or read book Ion Channels Channel Chemical Biology Engineering and Physiological Function written by and published by Academic Press. This book was released on 2021-06-12 with total page 454 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ion Channels, Part C, Volume 653 in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters on a variety of topics, including Nonsense suppression in ion channels, Engineering Ion Channels Using Protein Trans-splicing, Probing Ion Channel Neighborhoods Using APEX, STX based probes for NaVs, ANAP: a versatile, fluorescent probe of ion channel gating and regulation, High Throughput Screens for Small Molecule Ion Channel Modulators, Using toxins to study ion channels, Re/de-constructing ubiquitin regulation of ion channels, Tethered Peptide Toxins for Ion Channels, Voltage-Sensing Phosphatase Molecular Engineering, and more. Additional chapters cover Engineering excitable cells, Stretch and Poke Stimulation of Mechanically-Activated Ion Channels, Optical Control of STIM Channels, High Throughput Electrophysiological Evaluation of Mutant Ion Channels, Evaluating BEST1 Mutations in RPE Stem Cells, Long Read Transcript Profiling of Ion Channel Splice Variants, Permeation of Connexin Channels, Ratiometric pH indicator for melanosomes and lysosomes, and Ion channels in the epithelial cells of the choroid plexus. - Provides the authority and expertise of leading contributors from an international board of authors - Presents the latest release in the Methods in Enzymology series
Download or read book Decoding Neural Circuit Structure and Function written by Arzu Çelik and published by Springer. This book was released on 2017-07-24 with total page 517 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book offers representative examples from fly and mouse models to illustrate the ongoing success of the synergistic, state-of-the-art strategy, focusing on the ways it enhances our understanding of sensory processing. The authors focus on sensory systems (vision, olfaction), which are particularly powerful models for probing the development, connectivity, and function of neural circuits, to answer this question: How do individual nerve cells functionally cooperate to guide behavioral responses? Two genetically tractable species, mice and flies, together significantly further our understanding of these processes. Current efforts focus on integrating knowledge gained from three interrelated fields of research: (1) understanding how the fates of different cell types are specified during development, (2) revealing the synaptic connections between identified cell types (“connectomics”) using high-resolution three-dimensional circuit anatomy, and (3) causal testing of how iden tified circuit elements contribute to visual perception and behavior.
Download or read book Nuclear Architecture and Dynamics written by Christophe Lavelle and published by Academic Press. This book was released on 2017-10-27 with total page 620 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nuclear Architecture and Dynamics provides a definitive resource for (bio)physicists and molecular and cellular biologists whose research involves an understanding of the organization of the genome and the mechanisms of its proper reading, maintenance, and replication by the cell. This book brings together the biochemical and physical characteristics of genome organization, providing a relevant framework in which to interpret the control of gene expression and cell differentiation. It includes work from a group of international experts, including biologists, physicists, mathematicians, and bioinformaticians who have come together for a comprehensive presentation of the current developments in the nuclear dynamics and architecture field. The book provides the uninitiated with an entry point to a highly dynamic, but complex issue, and the expert with an opportunity to have a fresh look at the viewpoints advocated by researchers from different disciplines. - Highlights the link between the (bio)chemistry and the (bio)physics of chromatin - Deciphers the complex interplay between numerous biochemical factors at task in the nucleus and the physical state of chromatin - Provides a collective view of the field by a large, diverse group of authors with both physics and biology backgrounds
Download or read book Split Intein Applications for Downstream Purification and Protein Conjugation written by Jackelyn Galiardi and published by . This book was released on 2021 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Today, inteins have been extensively used in a wide variety of applications in biotechnology. Split inteins make up a small sector of inteins discovered more recently. Split inteins are expressed as two separate peptides naturally and are not active until intein-intein interaction. Due to their unique features, split inteins offer advanced controllability and flexibility in both self-cleaving and splicing activity. The natural split Npu intein was further engineered for pH controllability for applications including tag removal and on-column purification. The self-cleaving intein was first utilized as a tag removal platform for non-chromatographic aggregating tags. As a proof of concept, four target proteins were engineered with the aggregating beta-roll tag infusion with the pH controlled self-cleaving split intein. Beta-lactamase (beta-lac), super-folder green fluorescent protein (sfGFP), streptokinase (SK) and maltose binding protein (MBP), were successfully purified with the BRT17 intein fusion tag and produced tagless purified protein. The on-column purification strategy using the engineered self-cleaving split intein was employed for the production of valuable mammalian expressed target proteins and glycoprotein, ML39 single-chain variable fragments (scFv), Erythropoietin (EPO) and Interferon alpha-2b (IFN). All three protein were secreted, purified, and recovered at a high purity with a significant reduction of host-protein and DNA. Additionally, EPO and IFN glycosylation sites were further analyzed and verified. The use of nanotechnology in biomedical sciences has created a strong interest in its potential to dramatically improve the treatment and diagnosis of diseases. In collaboration with Columbus Nanoworks Inc, we developed simple and robust bioconjugation platforms for creating protein-labeled nanoparticles using split intein technology. In this work, we utilize two different split inteins, Gos-TerL and GP41.1, to achieve N-terminal and C-terminal oriented attachment of target proteins to nanoparticles, respectively. By mixing the peptide-labeled nanoparticles and fusion protein cell lysate, we have successfully demonstrated one-step bioconjugation of targets onto nanoparticles using the model proteins green fluorescent protein and beta-lactamase for both strategies.
