Heterologous Expression of Membrane Proteins


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.




Membrane Protein Protocols


Book Description

Knowledge of the three-dimensional structure of a protein is absolutely required for the complete understanding of its function. The spatial orientation of amino acids in the active site of an enzyme demonstrates how substrate specificity is defined, and assists the medicinal chemist in the design of s- cific, tight-binding inhibitors. The shape and contour of a protein surface hints at its interaction with other proteins and with its environment. Structural ana- sis of multiprotein complexes helps to define the role and interaction of each individual component, and can predict the consequences of protein mutation or conditions that promote dissociation and rearrangement of the complex. Determining the three-dimensional structure of a protein requires milligram quantities of pure material. Such quantities are required to refine crystallization conditions for X-ray analysis, or to overcome the sensitivity limitations of NMR spectroscopy. Historically, structural determination of proteins was limited to those expressed naturally in large amounts, or derived from a tissue or cell source inexpensive enough to warrant the use of large quantities of cells. H- ever, with the advent of the techniques of modern gene expression, many p- teins that are constitutively expressed in minute amounts can become accessible to large-scale purification and structural analysis.




Heterologous Gene Expression in E.coli


Book Description

Protein expression in a heterologous host is a cornerstone of biomedical research and of the biotechnology industry. Despite the advanced state of protein expression technology improvements are still needed. For example, membrane proteins constitute a significant percentage of the total cellular proteins but as a class are very difficult to overexpress, especially in a heterologous host. The ideal host would have the ability to express any protein, with relevant post-translational modifications, and be as easy to work with as E. coli. In Heterologous Gene Expression in E. coli: Methods and Protocols, expert scientists intimately familiar with the relevant techniques offer chapters that greatly expand the utility of this expression host. The contributions in this detailed volume describe methods, for example, to successfully express proteins in E. coli that would otherwise form aggregates in this host, to add post-translational modifications, to incorporate non-standard amino acid residues or moieties into E. coli expressed proteins, to identify binding partners, and to express membrane proteins. Written in the highly successful Methods in Molecular BiologyTM format, chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and cutting-edge, Heterologous Gene Expression in E. coli: Methods and Protocols seeks to familiarize the researcher with the myriad of E. coli expression strains available and move E. coli closer to that ideal of the perfect host.










Heterologous Gene Expression in E.coli


Book Description

This detailed volume provides a toolbox for designing constructs, tackling expression and solubility issues, handling membrane proteins and protein complexes, and exploring innovative engineering of E. coli. The topics are largely grouped under four parts: high-throughput cloning, expression screening, and optimization of expression conditions, protein production and solubility enhancement, case studies to produce challenging proteins and specific protein families, as well as applications of E. coli expression. 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 practical, Heterologous Gene Expression in E. coli: Methods and Protocols serves molecular biologists, biochemists and structural biologists, those in the beginning of their research careers to those in their prime, to give both an historical and modern overview of the methods available to express their genes of interest in this exceptional organism.




Membrane Protein Expression Systems


Book Description

The use of heterologous expression as a means to study membrane proteins is becoming widespread. This book is a simple to read laboratory manual describing the principles behind the commonly used expression systems, eg baculovirus, oocytes, cell culture systems and yeast.




Production of Membrane Proteins


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.




Membrane Proteins Production for Structural Analysis


Book Description

This book updates the latest development in production, stabilization and structural analysis techniques of membrane proteins. This field has made significant advances since the elucidation of the first 3-D structure of a recombinant G Protein Coupled Receptor (GPCR), rhodopsin, with the structure of several more GPCRs having been solved in the past five years. In fact, the 2012 Nobel Prize in Chemistry was awarded for groundbreaking discoveries on the inner workings of GPCRs. This book is essential reading for all researchers, biochemists and crystallographers working with membrane proteins, who are interested by the structural characterization of their favorite protein and who wish to follow the expression, migration, modifications and recycling of a membrane protein.




Plant ABC Transporters


Book Description

This book is devoted to the fascinating superfamily of plant ATP-binding cassette (ABC) transporters and their variety of transported substrates. It highlights their exciting biological functions, covering aspects ranging from cellular detoxification, through development, to symbiosis and defense. Moreover, it also includes a number of chapters that center on ABC transporters from non-Arabidopsis species. ABC proteins are ubiquitous, membrane-intrinsic transporters that catalyze the primary (ATP-dependent) movement of their substrates through biological membranes. Initially identified as an essential aspect of a vacuolar detoxification process, genetic work in the last decade has revealed an unexpectedly diverse variety of ABC transporter substrates, which include not only xenobiotic conjugates, but also heavy metals, lipids, terpenoids, lignols, alkaloids and organic acids. The discovery that members of the ABCB and ABCG family are involved in the movement of phytohormones has further sparked their exploration and provided a new understanding of the whole family. Accordingly, the trafficking, regulation and structure-function of ABCB-type auxin transporters are especially emphasized in this book.