Technology Development for the Over-expression, Purification and Crystallisation of Human Membrane Proteins PDF Download
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Author: Gillian Young
Publisher:
ISBN:
Category : Crystallization
Languages : en
Pages : 200
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Book Description
Author: Gillian Young
Publisher:
ISBN:
Category : Crystallization
Languages : en
Pages : 200
Get Book Here
Book Description
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Currently, the field of mammalian membrane protein structural biology is in its infancy. Existing technologies and experiences have shown that it is possible to obtain the structures of mammalian membrane proteins if sufficient work and thought has been invested. However, there is still an urgent need to develop new methodologies and approaches to improve all aspects of this important area of biological research. Here, a series of novel technologies for the overproduction, purification and crystallisation of human membrane proteins are described which have been tested with a representative member from each of the G-protein coupled receptor (adenosine 2a receptor (A2aR)) and membrane enzyme (sterol isomerase (SI)) superfamilies. The methylotrophic yeast Pichia pastoris is an excellent host cell for the overproduction of recombinant proteins including membrane proteins of mammalian origin. However, the commercially available expression vectors are far from what is required to maximise the production levels as well as simplify the detergent extraction and purification of human membrane proteins. Here, a series of related expression constructs were made that had different combinations of tags at both ends of the recombinant protein. The final optimised expression vectors had a C3 protease-iLOV-biotin acceptor-His10 (CLBH) tag fused to the C-terminus of the recombinant protein. The -CLBH vectors gave high level production of both test proteins (one Nin - hSI; one Nout - hA2aR) that could be rapidly purified to homogeneity using a generic protocol. The position of the His10 tag did not affect the expression level of the recombinant protein. In contrast, fusion of the biotin acceptor domain to the C-terminus of the recombinant protein increased its expression by a factor of between 2-4. The biotin acceptor domain could also be fully biotinylated in vitro using recombinantly expressed biotin ligase allowing purification/immobilisation of the target protein with streptavidin
Author: Hartmut Michel
Publisher: CRC Press
ISBN: 1351088173
Category : Medical
Languages : en
Pages : 343
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Book Description
The precise knowledge of the structure of biological macromolecules forms the basis of understanding their function and their mechanism of action. It also lays the foundation for rational protein and drug design. The only method to obtain this knowledge is still crystallography. At present, the structures of about 400 proteins are known at or nearly at atomic proteins. However, only two of them are membrane proteins or complexes of the membrane proteins. The reasons for the difference is not the crystals of membrane proteins resists forming special problems when being analysed. The reason is that the membrane proteins resist into forming into well-ordered crystals. The intention of this book is to help to produce well-ordered crystals proteins and to provide guidelines, it is aimed at both biochemists and protein crystallographer‘s.
Author:
Publisher: Academic Press
ISBN: 0080961592
Category : Science
Languages : en
Pages : 334
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Book Description
This volume of Current Topics in Membranes focuses on Membrane Protein Crystallization, beginning with a review of past successes and general trends, then further discussing challenges of mebranes protein crystallization, cell free production of membrane proteins and novel lipids for membrane protein crystallization. This publication also includes tools to enchance membrane protein crystallization, technique advancements, and crystallization strategies used for photosystem I and its complexes, establishing Membrane Protein Crystallization as a needed, practical reference for researchers.
Author: Carola Hunte
Publisher: Elsevier
ISBN: 0080536174
Category : Science
Languages : en
Pages : 339
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Book Description
This second edition of Membrane Protein Purification and Crystallization, A Practical Guide is written for bench scientists working in the fields of biochemistry, biology, and proteomic research. This guide presents isolation and crystallization techniques in a concise form, emphasizing the critical aspects unique to membrane proteins. It explains the principles of the methods and provides protocols of general use, permitting researchers and students new to this area to adapt these techniques to their particular needs. This edition is not only an update but is comprised mainly of new contributions. It is the first monograph compiling the essential approaches for membrane protein crystallization, and emphasizes recent progress in production and purification of recombinant membrane proteins. Provides general guidelines and strategies for isolation and crystallization of membrane proteins Gives detailed protocols that have wide application, and low specialized equipment needs Emphasizes recent progress in production and purification of recombinant membrane proteins, especially of histidine-tagged and other affinity-epitope-tagged proteins Summarizes recent developments of Blue-Native PAGE, a high resolution separation technique, which is independent of the use of recombinant techniques, and is especially suited for proteomic analyses of membrane protein complexes Gives detailed protocols for membrane protein crystallization, and describes the production and use of antibody fragments for high resolution crystallization Presents a comprehensive guide to 2D-crystallization of membrane proteins
Author: Anne Skaja Robinson
Publisher: John Wiley & Sons
ISBN: 3527634533
Category : Science
Languages : en
Pages : 631
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Book Description
Designed as a research-level guide to current strategies and methods of membrane protein production on the small to intermediate scale, this practice-oriented book provides detailed, step-by-step laboratory protocols as well as an explanation of the principles behind each method, together with a discussion of its relative advantages and disadvantages. Following an introductory section on current challenges in membrane protein production, the book goes on to look at expression systems, emerging methods and approaches, and protein specific considerations. Case studies illustrate how to select or sample the optimal production system for any desired membrane protein, saving both time and money on the laboratory as well as the technical production scale. Unique in its coverage of "difficult" proteins with large membrane-embedded domains, proteins from extremophiles, peripheral membrane proteins, and protein fragments.
Author: Sukarno Ferreira
Publisher: BoD – Books on Demand
ISBN: 9535110101
Category : Science
Languages : en
Pages : 436
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Book Description
Crystal growth is the key step of a great number of very important applications. The development of new devices and products, from the traditional microelectronic industry to pharmaceutical industry and many others, depends on crystallization processes. The objective of this book is not to cover all areas of crystal growth but just present, as specified in the title, important selected topics, as applied to organic and inorganic systems. All authors have been selected for being key researchers in their field of specialization, working in important universities and research labs around the world. The first section is mainly devoted to biological systems and covers topics like proteins, bone and ice crystallization. The second section brings some applications to inorganic systems and describes more general growth techniques like chemical vapor crystallization and electrodeposition. This book is mostly recommended for students working in the field of crystal growth and for scientists and engineers in the fields of crystalline materials, crystal engineering and the industrial applications of crystallization processes.
Author: Isabel Moraes
Publisher: Springer
ISBN: 3319350722
Category : Science
Languages : en
Pages : 188
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Book Description
This book reviews current techniques used in membrane protein structural biology, with a strong focus on practical issues. The study of membrane protein structures not only provides a basic understanding of life at the molecular level but also helps in the rational and targeted design of new drugs with reduced side effects. Today, about 60% of the commercially available drugs target membrane proteins and it is estimated that nearly 30% of proteins encoded in the human genome are membrane proteins. In recent years much effort has been put towards innovative developments to overcome the numerous obstacles associated with the structure determination of membrane proteins. This book reviews a variety of recent techniques that are essential to any modern researcher in the field of membrane protein structural biology. The topics that are discussed are not commonly found in textbooks. The scope of this book includes: Expression screening using fluorescent proteins The use of detergents in membrane protein research The use of NMR Synchrotron developments in membrane protein structural biology Visualisation and X-ray data collection of microcrystals X-ray diffraction data analysis from multiple crystals Serial millisecond crystallography Serial femtosecond crystallography Membrane protein structures in drug discovery The information provided in this book should be of interest to anyone working in the area of structural biology. Students will find carefully prepared overviews of basic ideas and advanced protein scientists will find the level of detail required to apply the material directly to their day to day work. Chapters 4, 5, 6, 8 and 9 of this book are published open access under a CC BY 4.0 license at link.springer.com.
Author: Isabelle Mus-Veteau
Publisher: Springer Nature
ISBN: 1071623680
Category : Science
Languages : en
Pages : 472
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Book Description
This detailed volume explores protocols for the production of membrane proteins in a panel of heterologous organisms for structural studies. Beginning with techniques using E. coli as a host for the overproduction and purification of membrane proteins, the book continues with chapters covering mammalian membrane protein production in yeast, insect cells, mammalian cells, as well as using virus like particles and acellular systems. Additionally, new detergents and alternatives to detergents allowing membrane protein purification for structural analyses are described. The book closes with a chapter exploring the use of microscale thermophoresis (MST) to evaluate the binding activity of heterologously expressed proteins directly in crude membrane extracts. 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, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Heterologous Expression of Membrane Proteins: Methods and Protocols, Third Edition serves as an ideal guide for scientists aiming to produce and purify functional recombinant membrane proteins for structural studies.
Author: Vivien Yeh
Publisher: Springer
ISBN: 9811312389
Category : Science
Languages : en
Pages : 139
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Book Description
This book focuses on the study of how the properties of nanodiscs, such as lipid composition and size, influence the function of the embedding integral membrane protein, bacteriorhodopsin. The author performed systematic studies to show that the lipid composition and the charge of the hydrophobic head and the structure of hydrophilic tails affect the photocycle pathway of bacteriorhodopsin, which is closely associated with its proton-pumping activity. Furthermore, the author demonstrated a highly efficient method for extracting membrane proteins directly from the biological membrane, preserving protein conformation, function and essential native lipids. This book demonstrates optimization and sample preparation, and presents practical methods of preparing membrane protein-embedded nanodisc samples for biophysical studies, which benefit structural and functional studies in the field of membrane protein characterization, both.
