Recombinant protein expression in microbial systems
Recombinant protein expression in microbial systems

Author: Eduardo A. Ceccarelli

Publisher: Frontiers E-books

Published: 2014-10-02

Total Pages: 103

ISBN-13: 2889192946

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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.

Molecular Biotechnology
Molecular Biotechnology

Author: Bernard R. Glick

Publisher:

Published: 1998

Total Pages: 724

ISBN-13:

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The second edition explains the principles of recombinant DNA technology as well as other important techniques such as DNA sequencing, the polymerase chain reaction, and the production of monclonal antibodies.

The Science and Applications of Synthetic and Systems Biology
The Science and Applications of Synthetic and Systems Biology

Author: Institute of Medicine

Publisher: National Academies Press

Published: 2011-12-30

Total Pages: 570

ISBN-13: 0309219396

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Many potential applications of synthetic and systems biology are relevant to the challenges associated with the detection, surveillance, and responses to emerging and re-emerging infectious diseases. On March 14 and 15, 2011, the Institute of Medicine's (IOM's) Forum on Microbial Threats convened a public workshop in Washington, DC, to explore the current state of the science of synthetic biology, including its dependency on systems biology; discussed the different approaches that scientists are taking to engineer, or reengineer, biological systems; and discussed how the tools and approaches of synthetic and systems biology were being applied to mitigate the risks associated with emerging infectious diseases. The Science and Applications of Synthetic and Systems Biology is organized into sections as a topic-by-topic distillation of the presentations and discussions that took place at the workshop. Its purpose is to present information from relevant experience, to delineate a range of pivotal issues and their respective challenges, and to offer differing perspectives on the topic as discussed and described by the workshop participants. This report also includes a collection of individually authored papers and commentary.

Production of Membrane Proteins
Production of Membrane Proteins

Author: Anne Skaja Robinson

Publisher: John Wiley & Sons

Published: 2011-06-15

Total Pages: 631

ISBN-13: 3527634533

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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.

Heterologous Gene Expression in E.coli
Heterologous Gene Expression in E.coli

Author: Nicola A. Burgess-Brown

Publisher: Humana Press

Published: 2018-07-20

Total Pages: 429

ISBN-13: 9781493983285

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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.

Molecular Biological Methods for Bacillus
Molecular Biological Methods for Bacillus

Author: Colin R. Harwood

Publisher: Wiley

Published: 1991-02-18

Total Pages: 618

ISBN-13: 9780471923930

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Molecular Biological Methods for Bacillus Edited by C. R. Harwood, Department of Microbiology, University of Newcastle upon Tyne, UK and S. M. Cutting, The Biological Laboratories, Harvard University, USA This volume represents the first major attempt to produce a compendium of the experimental methods used for the analysis of Bacillus, one of the most important procaryotic genera. Since the pioneering work on the transformation and genetic analysis of Bacillus subtilis by John Spizizen and his colleagues in the late 1950s this microorganism has been extensively studied and is now one of the best understood. More than forty of the world’s leading researchers in the field have generously contributed experimental procedures—devised and used in their laboratories—to this book. The aim throughout has been to present methods as simple step-by-step protocols which have been thoroughly tried and tested. The context in which the methods are used is discussed in detail and relevant information provided on the physiology and genetics of Bacillus. In addition valuable support is provided in the form of troubleshooting tips and advice on safety, the preparation of reagents, and the use of equipment. The book will be invaluable to those working with the genus Bacillus and related genera—both established researchers and those wishing to use this important microorganism for the first time.