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This volume provides an overview of the main yeast production platforms currently used and future yeast cell factories for recombinant protein production. Chapters detail approaches of genetic and metabolic engineering, co-factor containing proteins and virus-like particles, glycoproteins, and post-translational modifications of 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, Recombinant Protein Production in Yeast: Methods and Protocols aims to provide state of the art background and methods for protein producing yeast platforms, as well as case studies for special applications.
Since newly created beings are often perceived as either wholly good or bad, the genetic alteration of living cells impacts directly on a symbolic meaning deeply imbedded in every culture. During the earlier years of gene expression research, te- nological applications were confined mainly to academic and industrial laboratories, and were perceived as highly beneficial since molecules that were previously unable to be separated or synthesized became accessible as therapeutic agents. Such were the success stories of hormones, antibodies, and vaccines produced in the bacterium Escherichia coli. Originally this bacterium gained fame among humans for being an unwanted host in the intestine, or worse yet, for being occasionally dangerous and pathogenic. H- ever, it was easily identified in contaminated waters during the 19th century, thus becoming a clear indicator of water pollution by human feces. Tamed, cultivated, and easily maintained in laboratories, its fast growth rate and metabolic capacity to adjust to changing environments fascinated the minds of scientists who studied and modeled such complex phenomena as growth, evolution, genetic exchange, infection, survival, adaptation, and further on—gene expression. Although at the lower end of the complexity scale, this microbe became a very successful model system and a key player in the fantastic revolution kindled by the birth of recombinant DNA technology.
This book reviews preparation of expression vectors, generation of high-yielding clones, scale-up, disruption of yeast cells to enable isolation of recombinant protein prior to purification and more, in the popular Methods in Molecular Biology format."
Recombinant Protein Expression, Part B, Volume 660 in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters on Multiplexed analysis protein: Protein interactions of polypeptides translated in Leishmania cell-free system, MultiBac system and its applications, performance and recent, Production of antibodies in Shuffle, Designing hybrid-promoter architectures by engineering cis-acting DNA sites to enhance transcription in yeast, Designing hybrid-promoter architectures by engineering cis-acting DNA sites to deregulate transcription in yeast, Antibody or protein-based vaccine production in plants, Cell-free protein synthesis, Plant-based expression of biologic drugs, and much more. Additional sections cover the Use of native mass spectrometry to guide detergent-based rescue of non-native oligomerization by recombinant proteins, Advancing overexpression and purification of recombinant proteins by pilot optimization through tandem affinity-buffer exchange chromatography online with native mass spectrometry, Method for High-Efficiency Fed-batch cultures of recombinant Escherichia coli, Method to transfer Chinese hamster ovary (CHO) shake flask experiments to the ambr® 250, and Expression of recombinant antibodies in Leishmania tarentolae. - Provides the authority and expertise of leading contributors from an international board of authors - Presents the latest release in the Methods in Enzymology serial - Updated release includes the latest information on Recombinant Protein Expression
With the advent of recombinant DNA technology, expressing heterologous proteins in microorganisms rapidly became the method of choice for their production at laboratory and industrial scale. Bacteria, yeasts and other hosts can be grown to high biomass levels efficiently and inexpensively. Obtaining high yields of recombinant proteins from this material was only feasible thanks to constant research on microbial genetics and physiology that led to novel strains, plasmids and cultivation strategies. Despite the spectacular expansion of the field, there is still much room for progress. Improving the levels of expression and the solubility of a recombinant protein can be quite challenging. Accumulation of the product in the cell can lead to stress responses which affect cell growth. Buildup of insoluble and biologically inactive aggregates (inclusion bodies) lowers the yield of production. This is particularly true for obtaining membrane proteins or high-molecular weight and multi-domain proteins. Also, obtaining eukaryotic proteins in a prokaryotic background (for example, plant or animal proteins in bacteria) results in a product that lack post-translational modifications, often required for functionality. Changing to a eukaryotic host (yeasts or filamentous fungi) may not be a proper solution since the pattern of sugar modifications is different than in higher eukaryotes. Still, many advances in the last couple of decades have provided to researchers a wide variety of strategies to maximize the production of their recombinant protein of choice. Everything starts with the careful selection of the host. Be it bacteria or yeast, a broad list of strains is available for overcoming codon use bias, incorrect disulfide bond formation, protein toxicity and lack of post-translational modifications. Also, a huge catalog of plasmids allows choosing for different fusion partners for improving solubility, protein secretion, chaperone co-expression, antibiotic resistance and promoter strength. Next, controlling culture conditions like temperature, inducer and media composition can bolster recombinant protein production. With this Research Topic, we aim to provide an encyclopedic account of the existing approaches to the expression of recombinant proteins in microorganisms, highlight recent discoveries and analyze the future prospects of this exciting and ever-growing field.
This book focuses on recent developments of Pichia pastoris as a recombinant protein production system. Highlighted topics include a discussion on the use of fermentors to grow Pichia pastoris, information on the O- and N-linked glycosylation, methods for labeling Pichia pastoris expressed proteins for structural studies, and the introduction of mutations in Pichia pastoris genes by the methods of restriction enzyme-mediated integration (REMI). Each chapter presents cutting-edge and cornerstone protocols for utilizing P. pastoris as a model recomibinant protein production system. This volume fully updates and expands upon the first edition.
While the choices of microbial and eukaryotic expression systems for production of recombinant proteins are many, most researchers in academic and industrial settings do not have ready access to pertinent biological and technical information since it is normally scattered throughout the scientific literature. This book closes the gap by providing information on the general biology of the host organism, a description of the expression platform, a methodological section -- with strains, genetic elements, vectors and special methods, where applicable -- as well as examples of proteins produced with the respective platform. The systems thus described are well balanced by the inclusion of three prokaryotes (two Gram-negatives and one Gram-positive), four yeasts, two filamentous fungi and two higher eukaryotic cell systems -- mammalian and plant cells. Throughout, the book provides valuable practical and theoretical information on the criteria and schemes for selecting the appropriate expression platform, the possibility and practicality of a universal expression vector, and on comparative industrial-scale fermentation, with the production of a recombinant Hepatitis B vaccine chosen as an industrial example. With a foreword by Herbert P. Schweizer, Colorado State University, USA: "As a whole, this book is a valuable and overdue resource for a varied audience. It is a practical guide for academic and industrial researchers who are confronted with the design of the most suitable expression platform for their favorite protein for technical or pharmaceutical purposes. In addition, the book is also a valuable study resource for professors and students in the fields of applied biology and biotechnology."
Yeast Metabolic Engineering: Methods and Protocols provides the widely established basic tools used in yeast metabolic engineering, while describing in deeper detail novel and innovative methods that have valuable potential to improve metabolic engineering strategies in industrial biotechnology applications. Beginning with an extensive section on molecular tools and technology for yeast engineering, this detailed volume is not limited to methods for Saccharomyces cerevisiae, but describes tools and protocols for engineering other yeasts of biotechnological interest, such as Pichia pastoris, Hansenula polymorpha and Zygosaccharomyces bailii. Tools and technologies for the investigation and determination of yeast metabolic features are described in detail as well as metabolic models and their application for yeast metabolic engineering, while a chapter describing patenting and regulations with a special glance at yeast biotechnology closes the volume. Written in the highly successful Methods in Molecular Biology series format, most chapters include an introduction to their respective topic, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and authoritative, Yeast Metabolic Engineering: Methods and Protocols aims to familiarize researchers with the current state of these vital and increasingly useful technologies.
This book explores the journey of biotechnology, searching for new avenues and noting the impressive accomplishments to date. It has harmonious blend of facts, applications and new ideas. Fast-paced biotechnologies are broadly applied and are being continuously explored in areas like the environmental, industrial, agricultural and medical sciences. The sequencing of the human genome has opened new therapeutic opportunities and enriched the field of medical biotechnology while analysis of biomolecules using proteomics and microarray technologies along with the simultaneous discovery and development of new modes of detection are paving the way for ever-faster and more reliable diagnostic methods. Life-saving bio-pharmaceuticals are being churned out at an amazing rate, and the unraveling of biological processes has facilitated drug designing and discovery processes. Advances in regenerative medical technologies (stem cell therapy, tissue engineering, and gene therapy) look extremely promising, transcending the limitations of all existing fields and opening new dimensions for characterizing and combating diseases.
This book presents advanced expression technologies for the production of protein complexes. Since complexes lie at the heart of modern biology, the expression, purification, and characterization of large amounts of high-quality protein complexes is crucial for the fields of biomedicine, biotechnology, and structural biology. From co-expression in E. coli, yeast, mammalian and insect cells to complex reconstitution from individual subunits, this book offers useful insights and guidance for successful protein expressionists. Across several sections readers will discover existing opportunities for the production of protein complexes in bacterial systems (including membrane proteins and cell-free co-expression), methylotrophic and non-methylotrophic yeasts, protozoa (Leishmania terantolae and Dictyostelium discoideum), baculovirus-infected insect cells, mammalian cells, plants and algae. Complex reconstitution from individually purified subunits or subcomplexes is discussed as a complementary strategy. A last section introduces briefly some of the biophysical and structural characterization techniques for macromolecular complexes using state-of-the-art solution scattering and nuclear magnetic resonance. This work is a guided tour over some of the most powerful and successful protein expression technologies, with a focus on co-expression and high-throughput applications. It is addressed to everyone interested in the production and characterization of macromolecular complexes, from university students who want an accessible description of the major co-expression systems to researchers in biomedicine and the life sciences seeking for an up-to-date survey of available technologies.