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Baculovirus Expression Protocols offers both industrial and university-based researchers a comprehensive comilation of the latest baculovirus techniques along with step-by-step instructions and time-saving techniques. The contributors-leading authorities in the field-present the assorted expression plasmids currently in use, guide the reader through the process of generating and selecting recombinant virus, and describe specific examples of recombinant protein production and purification. The emphasis is on alternative and simpler screening techniques for the selection of recombinant baculovirus. The book also surveys the various insect cell lines currently compatible with the baculovirus system. Highlights include production of recombinant virus using linearized DNA and vectors that contain a b-galactosidase indicator and a complete list of expression vectors currently available. Detailed descriptions for the scale-up of protein production using spinner flasks, bioreactors, and insect larvae are also included. Baculovirus Expression Protocols offers both industrial and university-based researchers an outstanding collection of reproducible, step-by-step laboratory protocols. It will immediately become indispensable for anyone working with baculoviruses and their application in the expression of recombinant proteins in insect cells.
The third edition of this volume expands upon the previous two editions with new and up-to-date methods and protocols. Chapters include step-by-step procedures involved in quantifying cell growth, baculovirus infection and cell metabolism, methods to isolate new cell lines and develop your own serum-free medium, and routine maintenance and storage of insect cell lines and baculoviruses, small- and large-scale recombinant protein production with the BEVS in both insect and mammalian cell culture and in insect larvae, production and characterization of baculoviruses, green fluorescent protein, tubular reactors and RNAi, and baculovirus/insect cell system to study apoptosis and generating envelop-modified baculovirus for gene delivery into mammalian cells. 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 key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Baculovirus and Insect Cell Expression Protocols, Third Edition aims to not only aid the user in successfully completing the tasks described, but also stimulate the development of improved techniques and new applications of baculoviruses and insect cell culture.
Baculoviruses have proven to be the most powerful and versatile eukaryotic expression vectors available. This unique laboratory manual is designed to help both beginning and experienced researchers construct and use baculovirus vector systems. It simplifies selection of the most appropriatebaculovirus vector design for a given problem, then describes each step of the implementation process--from vector construction to large-scale protein production. The book provides an understanding of how the vectors work; a biological overview of cells, viruses, plasmids, and promoters; guidelinesfor choosing optimum vectors; protocols for growing insect cells and recombinant viruses; methods of analyzing protein products and scaling up protein production; techniques for producing proteins in insect larvae; and easy-to-use maps charting available expression vectors. This comprehensiveapproach has many benefits for researchers and students alike. It allows them to understand how and why the vector system works and offers a rapid comparison of options for choosing the right virus, plasmid or promoter for vector design and construction, with a minimum amount of lost time. Themanual is an invaluable resource for every individual engaged in the production of proteins for any purpose.
"Baculovirus Expression Protocols" is a detailed guide for using the baculovirus expression vector system (BEVS) and/or insect cells to produce recombinant proteins. Procedures for the development and production of baculovirus insecticides at both laboratory and large scale are described in the kind of step-by-step format that the "Methods in Molecular Biology" series helped standardize. This book is a one-stop source for information on baculoviruses, and includes sections on using modified baculoviruses to express genes in mammalian cells, using Drosophila cell lines, isolating new cell lines, and developing serum-free medium. Specific applications, including using baculovirus and insect cell system to study apoptosis, are also discussed. This updated and expanded edition of "Baculovirus Expression Protocols" provides a perfect lab manual for biochemists, molecular biologists, biochemical engineers, and others for using the BEVS or insect cell lines to produce recombinant proteins.
The decision to write a book about the practical aspects of the baculovirus expression system stems from the numerous phone calls for help we have had, and from the many visitors to our labora tories requiring assistance to find the elusive polyhedrin-negative virus containing their favourite gene. We have also organized two expression system workshops and from the manuals we wrote for these, it seemed a logical progression to extend them into book form. We appreciate that those who are 'old-hands' at the baculovirus expression system may have differing views on some of our procedures, but the methods in this book are presented in the light of our own experiences in the laboratory and from our practical workshops, and we hope that the book will be especially useful to those new to the system. The first three chapters give the background information to the baculovirus expression system, and includes advice on how to choose the right transfer vector and discusses the various methods that are available to select recombinant viruses. The practical chapters concentrate on those aspects which are novel to the baculovirus system (insect cell culture, virus amplification and titration, etc. ) and, in general, leave the standard molecular biological techniques to the other excellent laboratory manuals that are available. However, for completeness sake and to avoid constant reference to other manuals, we have included brief details of some standard techniques where they are integral to the success of the baculovirus protocols.
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
1. Expression strategy (Michael Dyson) 2. Protein expression in Escherichia coli (Rosalind Kim) 3. Expression engineering of synthetic antibodies using ribosome display (Matthew DeLisa and Lydia M. Contreras Martinez) 4. Refolding proteins from inclusion bodies (Renaud Vincentelli) 5. Selection of protein variants with improved expression using GFP-derived folding and solubility reporters (Geoffrey Waldo and Stéphanie Cabantous) 6. Protein expression in the wheat germ cell-free system (Yaeta Endo and Tatsuya Sawasaki) 7. Saccharomyces cerevisiae ; A microbial eukaryotic expression system (Christine Lang) 8. Expression of proteins in Pichia pastoris (Geoff and Joan Lin-Cereghino and Wilson Leung) 9. Improved baculovirus expression vectors (Linda King, Richard Hitchman and Robert Possee) 10. Transient transfection of insect cells for rapid expression screening and protein production (Robert Novy et al.) 11. Generation of stable CHO cell lines for protein expression (Zhijian Lu et al.) 12. Transient expression in HEK293-EBNA1 cells (Yves Durocher, Roseanne Tom and Louis Bisson) 13. Nisin- and subtilin-controlled gene expression systems for Gram-positive bacteria (Oscar Kuipers and Jan Kok) 14. Protein expression using lentiviral vectors (Bernard Massie, Renald Gilbert and Sophie Broussau) 15. Expression in mammalian cells using BacMam viruses (Yu-Chen Hu and Hsiao-Ping Lee) List of suppliers;Index
Baculovirus Expression Protocols, Second Edition, provides the detailed steps required to perform the techniques involved with the use of baculoviruses and insect cell culture and discusses problems that may be encountered. This newly-revised and updated edition provides a cutting edge step-by-step guide for biochemists, molecular biologists, biochemical engineers and others using the BEVS and/or insect cells for producing recombinant proteins. Furthermore, Baculovirus Expression Protocols, Second Edition, provides assistance to scientists and engineers interested in developing and producing baculovirus insecticides. Authors list all available insect cell lines and provide methods for isolating new cell lines. The procedures involved in producing products, both at laboratory scale and large scale, are discussed, as well as the production in insect larvae. It is hoped that this volume will not only aid the user in successfully completing the tasks described herein, but will also stimulate the development of improved techniques and new applications of baculoviruses and insect cell culture. Timely and thorough, this volume will become an essential reference for anyone using baculoviruses and insect cell culture.