Enzyme-Mediated Ligation Methods - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Enzyme-Mediated Ligation Methods write by Timo Nuijens. This book was released on 2019-06-03. Enzyme-Mediated Ligation Methods available in PDF, EPUB and Kindle. This volume discusses different enzyme-catalyzed ligation methodologies for a variety of different chemical transformations. This book wants readers to view enzymes as a powerful tool in both academic and industrial research. Chapters in this book cover topics such as sortase A-mediated generation of site-specifically conjugated antibody-drug conjugates; omniligase-catalyzed inter- and intramolecular ligation; ligation catalyzed by microbial transglutaminase; peptide cyclization mediated by cyanobactin macrocyclases, butelase 1 and sortase A; using BioID as a tool for protein proximity labeling in living cells; and inducible, selective labeling of proteins via enzymatic oxidation of tyrosine. 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. Cutting-edge and thorough, Enzyme-Mediated Ligation Methods is a valuable resource for students and scientists from different disciplines who are interested in using enzymatic strategies to answer their research questions.
Enzyme-mediated Labeling of Proteins and Protein-protein Interactions in Vitro and in Living Cells
Enzyme-mediated Labeling of Proteins and Protein-protein Interactions in Vitro and in Living Cells - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Enzyme-mediated Labeling of Proteins and Protein-protein Interactions in Vitro and in Living Cells write by Sarah Ann Slavoff. This book was released on 2010. Enzyme-mediated Labeling of Proteins and Protein-protein Interactions in Vitro and in Living Cells available in PDF, EPUB and Kindle. The E. coli biotin ligase enzyme, BirA, has been previously used by the Ting research group for site-specific labeling of peptide-tagged cell surface proteins. We sought to expand the utility of biotin ligase-mediated labeling to functional group handles, including azides and alkynes, for bio-orthogonal chemistry. Since the BirA and its point mutants were unable to ligate these probes to an acceptor peptide, we screened biotin ligases from multiple species to identify more permissive enzymes. We determined that the Pyrococcus horikoshii biotin ligase utilizes an azide-bearing biotin analog and that the Saccharomyces cerevisiae biotin ligase can utilize an alkyne-functionalized biotin analog. We subsequently demonstrated that the azidefunctionalized biotin analog can be derivatized with a phosphine probe via the Staudinger ligation. We next turned to the goal of delivering quantum dots to the cytosol of living cells, which in the future may permit intracellular single-molecule imaging. We investigated viral methods of delivery, but found that our protocol caused quantum dots to be trapped in endocytic vesicles. We then validated previous reports that the pore-forming toxin streptolysin 0 be used to deliver quantum dots to the cytosol of living cells. Lipoic acid ligase, or LpIA, has been previously applied to site-specific protein labeling of peptide-tagged proteins using small molecule probes including lipoic acid and coumarin fluorophores. We utilized LpIA and its substrate, the LAP peptide, to create sensors for proteinprotein interactions. If LpIA is fused to one protein and LAP is fused to another, only when the two proteins interact do LpIA and LAP come into proximity, allowing probe ligation onto the peptide to occur as a readout of the interaction. We demonstrate that proximity-dependent coumarin ligation detects protein-protein interactions in living mammalian cells with extremely low background, a signal-to-background ratio of at least 5:1, and sufficiently fast kinetics to label interactions with a half-life of at least 1 minute. The reporter quantitatively responds to subpopulations of interacting proteins, allowing dissociation constants to be measured. Coumarin fluorescence accurately reports the subcellular localization of the interaction under study. Finally, we applied proximity-dependent coumarin ligation to imaging of the interaction of PSD-95 and neuroligin-1, two proteins involved in synaptic maturation, in neurons.
Driving Sortase-mediated Ligations Using Metal-coordinating Peptides
Driving Sortase-mediated Ligations Using Metal-coordinating Peptides - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Driving Sortase-mediated Ligations Using Metal-coordinating Peptides write by Sierra Reed. This book was released on 2018. Driving Sortase-mediated Ligations Using Metal-coordinating Peptides available in PDF, EPUB and Kindle. The versatility of sortase-mediated ligations as a protein modification technique has been well demonstrated, but the efficiency of these reactions suffers from inherent reversibility. Solutions to this issue have been reported, however these methods are accompanied by additional limitations of the sortase-mediated ligation (SML) strategy. A preferable methodology would include the smallest possible modification site without restricting the point of ligation. One promising solution to this issue is the expansion of the LPXTG SrtA recognition sequence to LPXTGGH, giving the excised fragment an N-terminal GGH motif. This minor alteration has been shown to allow complexation of the excised fragment with Ni2+ ions, thus sequestering this component from the reaction and improving yields through hindered reversibility. In this thesis, we explore the scope of this metal-assisted sortase-mediated ligation (MASML) approach, including the Ni2+-enhanced modification of full sized proteins with a number of useful chromophores. Furthermore, this approach was shown to be compatible with the installation of PEG and a cyclooctyne bioorthogonal ligation handle. In total, this work demonstrates that MASML is compatible with a range of high value protein targets and modifications, and shows how MASML is a straightforward method for improving the efficiency of sortase-mediated protein engineering strategies.
Plant Cyclotides
Plant Cyclotides - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Plant Cyclotides write by . This book was released on 2015-11-24. Plant Cyclotides available in PDF, EPUB and Kindle. 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
Chemoselective and Bioorthogonal Ligation Reactions
Chemoselective and Bioorthogonal Ligation Reactions - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Chemoselective and Bioorthogonal Ligation Reactions write by W. Russ Algar. This book was released on 2017-06-19. Chemoselective and Bioorthogonal Ligation Reactions available in PDF, EPUB and Kindle. This timely, one-stop reference is the first on an emerging and interdisciplinary topic. Covering both established and recently developed ligation chemistries, the book is divided into two didactic parts: a section that focuses on the details of bioorthogonal and chemoselective ligation reactions at the level of fundamental organic chemistry, and a section that focuses on applications, particularly in the areas of chemical biology, biomaterials, and bioanalysis, highlighting the capabilities and benefits of the ligation reactions. With chapters authored by outstanding scientists who range from trailblazers in the field to young and emerging leaders, this book on a highly interdisciplinary topic will be of great interest for biochemists, biologists, materials scientists, pharmaceutical chemists, organic chemists, and many others.
