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Emerging Biotechnology to Detect Weak And/or Transient Protein-protein Interactions

Emerging Biotechnology to Detect Weak And/or Transient Protein-protein Interactions
Author: Gabrielle Nina Thibodeaux
Publisher:
Total Pages: 310
Release: 2009
Genre:
ISBN:

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Protein-protein interactions are of great importance to a number of essential biological processes including cell cycle regulation, cell-cell interactions, DNA replication, transcription and translation. Thus, an understanding of protein-protein interactions is critical for understanding many facets of cell function. Unfortunately, the tools and methods currently in use to identify and study protein-protein interactions focus largely on high affinity, stable interactions. However, the majority of the protein-protein interactions involved in regulatory processes have weak affinities and are transient in nature. Therefore, it is important to develop new biotechnology capable of detecting weak and/or transient protein-protein interactions in vivo. Here, we describe four new methods that allow for the identification and study of weak and/or transient protein-protein interactions in vivo. First, we developed a rapid method to convert Escherichia coli orthogonal tRNA/synthetase pairs into an orthogonal system for mammalian cells in order to site-specifically incorporate unnatural amino acids into any gene of interest using stop codon suppression. This method will allow the expression and purification of proteins that carry normally transient post-translational modifications. Second, we successfully employed site-specific unnatural amino acid incorporation to chemically cross-link a known homodimer, Sortase A, in vivo. Third, we developed a novel tetracycline repressor-based mammalian two-hybrid system and successfully detected homo- and hetero-dimers that are known to have weak binding constants. Finally, a synthetic antibody (termed a synbody) that binds weakly to the SH3 domain of the proto-oncogene Abelson tyrosine kinase was developed. The synbody can potentially be used as a first generation drug and/or biomarker. We hope that the methods developed in this dissertation will enable the scientific community to better understand weak/transient protein-protein interactions in vivo.


Studies in Pharmaceutical Biotechnology

Studies in Pharmaceutical Biotechnology
Author: Aiko Umeda
Publisher:
Total Pages: 376
Release: 2011
Genre:
ISBN:

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Pharmaceutical biotechnology has been emerging as a defined, increasingly important area of science dedicated to the discovery and delivery of drugs and therapies for the treatment of various human diseases. In contrast to the advancement in pharmaceutical biotechnology, current drug discovery efforts are facing unprecedented challenges. Difficulties in identifying novel drug targets and developing effective and safe drugs are closely related to the complexity of the network of interacting human proteins. Protein-protein interactions mediate virtually all cellular processes. Therefore both identification and understanding of protein-protein interactions are essential to the process of deciphering disease mechanisms and developing treatments. Unfortunately, our current knowledge and understanding of the human interactome is largely incomplete. Most of the unknown protein-protein interactions are expected to be weak and/or transient, hence are not easily identified. These unknown or uncharacterized interactions could affect the efficacy and toxicity of drug candidates, contributing to the high rate of failure. In an attempt to facilitate the ongoing efforts in drug discovery, we describe herein a series of novel methods and their applications addressing the broad topic of protein-protein interactions. We have developed a highly efficient site-specific protein cross-linking technology mediated by the genetically incorporated non-canonical amino acid L-DOPA to facilitate the identification and characterization of weak protein-protein interactions. We also established a protocol to incorporate L-DOPA into proteins in mammalian cells to enable in vivo site-specific protein cross-kinking. We then applied the DOPA-mediated cross-linking methodology to design a protein probe which can potentially serve as a diagnostic tool or a modulator of protein-protein interactions in vivo. To deliver such engineered proteins or other bioanalytical reagents into single live cells, we established a laser-assisted cellular nano-surgery protocol which would enable detailed observations of cell-to-cell variability and communication. Finally we investigated a possible experimental scheme to genetically evolve a fluorescent peptide, which has tremendous potential as a tool in cellular imaging and dynamic observation of protein-protein interactions in vivo. We aim to contribute to the discovery and development of new drugs and eventually to the overall health of our society by adding the technology above to the array of currently available bioanalytical tools.


Proteins

Proteins
Author: Thomas E. Creighton
Publisher: Macmillan
Total Pages: 534
Release: 1993
Genre: Medical
ISBN: 9780716770305

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Organized on a combined basis of chronology and of structural and functional hierarchy, This comprehensive text describes all aspects of proteins--biosynthesis, evolution, dynamics, ligand binding, catalysis, and energy transduction--not just their structures. This edition (first was 1984) is thoroughly updated--especially in the area of protein biosynthesis--and features end-of-chapter exercises and problems, many of which require the student to consult the cited literature in order to obtain the answer. Annotation copyright by Book News, Inc., Portland, OR


The Yeast Two-hybrid System

The Yeast Two-hybrid System
Author: Paul L. Bartel
Publisher: Oxford University Press, USA
Total Pages: 362
Release: 1997
Genre: Carrier proteins
ISBN: 9780195109382

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This volume, part of the Advances in Molecular Biology series, presents work by pioneers in the field and is the first publication devoted solely to the yeast two-hybrid system. It includes detailed protocols, practical advice on troubleshooting, and suggestions for future development. In addition, it illustrates how to construct an activation domain hybrid library, how to identify mutations that disrupt an interaction, and how to use the system in mammalian cells. Many of the contributors have developed new applications and variations of the technique.


Molecular Biology of The Cell

Molecular Biology of The Cell
Author: Bruce Alberts
Publisher:
Total Pages: 0
Release: 2002
Genre: Cytology
ISBN: 9780815332183

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Abiotic Stress-Mediated Sensing and Signaling in Plants: An Omics Perspective

Abiotic Stress-Mediated Sensing and Signaling in Plants: An Omics Perspective
Author: Sajad Majeed Zargar
Publisher: Springer
Total Pages: 358
Release: 2018-02-20
Genre: Science
ISBN: 9811074798

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The natural environment for plants is composed of a complex set of abiotic and biotic stresses; plant responses to these stresses are equally complex. Systems biology allows us to identify regulatory hubs in complex networks. It also examines the molecular “parts” (transcripts, proteins and metabolites) of an organism and attempts to combine them into functional networks or models that effectively describe and predict the dynamic activities of that organism in different environments. This book focuses on research advances regarding plant responses to abiotic stresses, from the physiological level to the molecular level. It highlights new insights gained from the integration of omics datasets and identifies remaining gaps in our knowledge, outlining additional focus areas for future crop improvement research. Plants have evolved a wide range of mechanisms for coping with various abiotic stresses. In many crop plants, the molecular mechanisms involved in a single type of stress tolerance have since been identified; however, in order to arrive at a holistic understanding of major and common events concerning abiotic stresses, the signaling pathways involved must also be elucidated. To date several molecules, like transcription factors and kinases, have been identified as promising candidates that are involved in crosstalk between stress signalling pathways. However, there is a need to better understand the tolerance mechanisms for different abiotic stresses by thoroughly grasping the signalling and sensing mechanisms involved. Accordingly, this book covers a range of topics, including the impacts of different abiotic stresses on plants, the molecular mechanisms leading to tolerance for different abiotic stresses, signaling cascades revealing cross-talk among various abiotic stresses, and elucidation of major candidate molecules that may provide abiotic stress tolerance in plants.


Preparing for Future Products of Biotechnology

Preparing for Future Products of Biotechnology
Author: National Academies of Sciences, Engineering, and Medicine
Publisher: National Academies Press
Total Pages: 231
Release: 2017-07-28
Genre: Science
ISBN: 0309452058

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Between 1973 and 2016, the ways to manipulate DNA to endow new characteristics in an organism (that is, biotechnology) have advanced, enabling the development of products that were not previously possible. What will the likely future products of biotechnology be over the next 5â€"10 years? What scientific capabilities, tools, and/or expertise may be needed by the regulatory agencies to ensure they make efficient and sound evaluations of the likely future products of biotechnology? Preparing for Future Products of Biotechnology analyzes the future landscape of biotechnology products and seeks to inform forthcoming policy making. This report identifies potential new risks and frameworks for risk assessment and areas in which the risks or lack of risks relating to the products of biotechnology are well understood.


Protein-Protein Interactions

Protein-Protein Interactions
Author: Weibo Cai
Publisher: BoD – Books on Demand
Total Pages: 488
Release: 2012-03-30
Genre: Science
ISBN: 9535103970

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Proteins are indispensable players in virtually all biological events. The functions of proteins are coordinated through intricate regulatory networks of transient protein-protein interactions (PPIs). To predict and/or study PPIs, a wide variety of techniques have been developed over the last several decades. Many in vitro and in vivo assays have been implemented to explore the mechanism of these ubiquitous interactions. However, despite significant advances in these experimental approaches, many limitations exist such as false-positives/false-negatives, difficulty in obtaining crystal structures of proteins, challenges in the detection of transient PPI, among others. To overcome these limitations, many computational approaches have been developed which are becoming increasingly widely used to facilitate the investigation of PPIs. This book has gathered an ensemble of experts in the field, in 22 chapters, which have been broadly categorized into Computational Approaches, Experimental Approaches, and Others.


Handbook of Molecular Biotechnology

Handbook of Molecular Biotechnology
Author: Dongyou Liu
Publisher: CRC Press
Total Pages: 763
Release: 2024-09-05
Genre: Science
ISBN: 1040005640

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With a history that likely dates back to the dawn of human civilization more than 10,000 years ago, and a record that includes the domestication and selective breeding of plants and animals, the harnessing of fermentation process for bread, cheese, and brewage production, and the development of vaccines against infectious diseases, biotechnology has acquired a molecular focus during the 20th century, particularly following the resolution of DNA double helix in 1953, and the publication of DNA cloning protocol in 1973, and transformed our concepts and practices in disease diagnosis, treatment and prevention, pharmaceutical and industrial manufacturing, animal and plant industry, and food processing. While molecular biotechnology offers unlimited opportunities for improving human health and well-being, animal welfare, agricultural innovation and environmental conservation, a dearth of high quality books that have the clarity of laboratory manuals without distractive procedural details and the thoroughness of well-conversed textbooks appears to dampen the enthusiasm of aspiring students. In attempt to fill this glaring gap, Handbook of Molecular Biotechnology includes four sections, with the first three presenting in-depth coverage on DNA, RNA and protein technologies, and the fourth highlighting their utility in biotechnology. Recognizing the importance of logical reasoning and experimental verification over direct observation and simple description in biotechnological research and development, the Introduction provides pertinent discussions on key strategies (i.e., be first, be better, and be different), effective thinking (lateral, parallel, causal, reverse, and random), and experimental execution, which have proven invaluable in helping advance research projects, evaluate and prepare research reports, and enhance other scientific endeavors. Key features Presents state-of-the-art reviews on DNA, RNA and protein technologies and their biotechnological applications Discusses key strategies, effective thinking, and experimental execution for scientific research and development Fills the gap left by detailed-ridden laboratory manuals and insight-lacking standard textbooks Includes expert contributions from international scientists at the forefront of molecular biotechnology research and development Written by international scientists at the forefront of molecular biotechnology research and development, chapters in this volume cover the histories, principles, and applications of individual techniques/technologies, and constitute stand-alone, yet interlinked lectures that strive to educate as well as to entertain. Besides providing an informative textbook for tertiary students in molecular biotechnology and related fields, this volume serves as an indispensable roadmap for novice scientists in their efforts to acquire innovative skills and establish solid track records in molecular biotechnology, and offers a contemporary reference for scholars, educators, and policymakers wishing to keep in touch with recent developments in molecular biotechnology.


Protein-Protein Interaction Networks

Protein-Protein Interaction Networks
Author: Stefan Canzar
Publisher: Humana
Total Pages: 0
Release: 2019-10-04
Genre: Science
ISBN: 9781493998722

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This volume explores techniques that study interactions between proteins in different species, and combines them with context-specific data, analysis of omics datasets, and assembles individual interactions into higher-order semantic units, i.e., protein complexes and functional modules. The chapters in this book cover computational methods that solve diverse tasks such as the prediction of functional protein-protein interactions; the alignment-based comparison of interaction networks by SANA; using the RaptorX-ComplexContact webserver to predict inter-protein residue-residue contacts; the docking of alternative confirmations of proteins participating in binary interactions and the visually-guided selection of a docking model using COZOID; the detection of novel functional units by KeyPathwayMiner and how PathClass can use such de novo pathways to classify breast cancer subtypes. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary hardware- and software, step-by-step, readily reproducible computational protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Protein-Protein Interaction Networks: Methods and Protocols is a valuable resource for both novice and expert researchers who are interested in learning more about this evolving field.