Author: John Alan WillisPublisher:
ISBN:
Category :
Languages : en
Pages : 0
Book Description
The Optimization of Streamlined Expressed Protein Ligation for Antibodies
Author: John Alan WillisThe Development, Use, and Optimization of Sortase-tag Expressed Protein Ligation
Author: Robert L. Warden-RothmanPublisher:
ISBN:
Category :
Languages : en
Pages : 348
Book Description
Molecular imaging is an emerging field that seeks to combine the mechanistic detail of biochemical assays with the the broad phenotypic data obtained from non-invasive medical imaging. A cornerstone of molecular imaging is the ability to chemically attach a contrast-inducing payload to a targeting ligand, a protein that binds tightly and specifically to the targeted cell population. There are currently many approaches to bioconjugation. Chemical approaches have broad applicability, but lack the site-specificity and efficiency desired for contrast agent generation. Enzymatic techniques are quantitative, efficient, and site-specific, but often have limited scope and can add significant bulk to the targeting ligand or require additional downstream purification steps. In this work, we create a novel bioconjugation and protein purification technique, Sortase-Tag Expressed Protein Ligation (STEPL). By fusing the active domain of S. aureus Sortase A to the targeting ligand as part of a greater chimeric protein, we are able to combine protein purification and the C-terminal bioconjugation of that protein into a single step. Using a mass-action kinetics model, we prove that this results in a modular, efficient system capable of producing high-purity conjugated protein. We then use the system to generate contrast agents for fluorescent and magnetic imaging studies. The modularity of the STEPL system allows us to create an array of monospecific and bispecific targeting ligand dimers, linked by two different spacer regions. The dimers' binding properties are probed and they are used to successfully bind and label cells, with the bispecific proteins enhancing contrast of a cell line that is positive for both targets. Finally the STEPL system is subjected to a number of directed evolution approaches and screened for a clone that could further improve STEPL's efficiency and yield.
Expressed Protein Ligation
Author: Miquel Vila-PerellóPublisher: Humana
ISBN: 9781071604335
Category : Science
Languages : en
Pages : 0
Book Description
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.
Protein Semisynthesis and Engineering by Expressed Protein Ligation
Author: Zuzana MachovaNon-Natural Amino Acids
Author: Publisher: Academic Press
ISBN: 0080921639
Category : Science
Languages : en
Pages : 334
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
Split Inteins
Author: Henning D. MootzPublisher: Humana
ISBN: 9781493964499
Category : Science
Languages : en
Pages : 0
Book Description
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.
Innovations for Next-Generation Antibody-Drug Conjugates
Author: Marc DamelinPublisher: Springer
ISBN: 3319781545
Category : Medical
Languages : en
Pages : 358
Book Description
Antibody-drug conjugates (ADCs) stand at the verge of a transformation. Scores of clinical programs have yielded only a few regulatory approvals, but a wave of technological innovation now empowers us to overcome past technical challenges. This volume focuses on the next generation of ADCs and the innovations that will enable them. The book inspires the future by integrating the field’s history with novel strategies and cutting-edge technologies. While the book primarily addresses ADCs for solid tumors, the last chapter explores the emerging interest in using ADCs to treat other diseases. The therapeutic rationale of ADCs is strong: to direct small molecules to the desired site of action (and away from normal tissues) by conjugation to antibodies or other targeting moieties. However, the combination of small and large molecules imposes deep complexity to lead optimization, pharmacokinetics, toxicology, analytics and manufacturing. The field has made significant advances in all of these areas by improving target selection, ADC design, manufacturing methods and clinical strategies. These innovations will inspire and educate scientists who are designing next-generation ADCs with the potential to transform the lives of patients.
Cell-Free Protein Expression
Author: James R. SwartzPublisher: Springer Science & Business Media
ISBN: 3642593372
Category : Science
Languages : en
Pages : 213
Book Description
Cell-free protein synthesis is coming of age! Motivated by an escalating need for efficient protein synthesis and empowered by readily accessible cell-free protein synthesis kits, the technology is expanding both in the range of feasible proteins and in the ways that proteins can be labeled and modified. This volume follows "Cell-Free Translation Systems", edited by Professor Alexander S. Spirin in 2002. Since then, an impressive collection of new work has emerged that demonstrates a substantial expansion of capability. In this volume, we show that proteins now can be efficiently produced using PCR products as DNA templates and that even membrane proteins and proteins with multiple disulfide proteins are obtained at high yields. Many additional advances are also presented. It is an exciting time for protein synthesis technology.
Human Monoclonal Antibodies
Author: Michael SteinitzPublisher: Humana Press
ISBN: 9781627035859
Category : Medical
Languages : en
Pages : 0
Book Description
The introduction of monoclonal antibodies revolutionized immunology. The development of human monoclonal antibodies was inspired primarily by the enormous clinical benefits promised by these reagents which can be used as anti-inflammatory reagents, anti-tumor reagents and reagents for passive immunization in a variety of pathologies. Human Monoclonal Antibodies: Methods and Protocols presents technical protocols of cellular and molecular methods for the production, purification and application of human monoclonal antibodies, as well as review articles on related topics of human monoclonal and polyclonal antibodies. Written in the 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 protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Human Monoclonal Antibodies: Methods and Protocols seeks to serve both professionals and novices with its well-honed methodologies which will prove invaluable in a clinical setting.
Molecular Epidemiology
Author: Paul A. SchultePublisher: Academic Press
ISBN: 0323138578
Category : Medical
Languages : en
Pages : 609
Book Description
This book will serve as a primer for both laboratory and field scientists who are shaping the emerging field of molecular epidemiology. Molecular epidemiology utilizes the same paradigm as traditional epidemiology but uses biological markers to identify exposure, disease or susceptibility. Schulte and Perera present the epidemiologic methods pertinent to biological markers. The book is also designed to enumerate the considerations necessary for valid field research and provide a resource on the salient and subtle features of biological indicators.