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 appropriate baculovirus 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; guidelines for 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 comprehensive approach 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. The manual is an invaluable resource for every individual engaged in the production of proteins for any purpose.

Acknowledgments How To Use This Manual PART I: AN OVERVIEW OF BACULOVIRUSES 1. Virus Structure and the Infection Process 2. Gene Organization, Regulation, and Function 3. Virus-Host Interactions 4. Summary of Baculovirus Features Relevant to Expression Factors PART II: CHOOSING A TRANSFER PLASMID AND PARENT VIRUS 5. Choice of Virus and Host Species 6. Choice of Transfer Plasmid 7. Available Transfer Plasmids 8. Choosing a Parent Virus for Use in Vector Construction 9. Optimizing Expression: Tailoring the Heterologous Gene to the Transfer Plasmid and the Baculovirus Expression System PART III: METHODS FOR VECTOR CONSTRUCTION AND GENE EXPRESSION 10. Safety Considerations 11. Insect Cell Culture 12. Virus Methods 13. Contransfection and Recombinant Virus Identification 14. Characterizing Recombinant Gene Expression 15. Post-Translational Modification PART IV: METHODS FOR SCALE-UP OF PROTEIN PRODUCTION AND USE OF INSECT LARVAE 16. Scale-Up of Protein Production in a Stirred Bioreactor, David L. Clemm 17. Scale-Up of Protein Production in an Airlift Fermenter, Cheryl Isaac Murphy 18. Insect Rearing and Infection Appendix 1: Nucleotide Sequence of AcMNPV Polyhedrin Region Appendix 2: Nucleotide Sequence of AcMNPV p10 Region Appendix 3: List of Genes Expressed in the Baculovirus Expression System Appendix 4: Suppliers Appendix 5: Stock Solutions Appendix 6: Excel Spreadsheet for TCID50 Calculation References Index

David R. O'ReillyLecturer in Cell Biology, Department of Biology, Imperial College, London, Lois K. MillerProfessor, Departments of Entomology and Genetics, University of Georgia, Verne A. LuckowResearch Scientist, Monsanto Corporation

"A dedicated monograph. . . . a brief but thorough and well-referenced review . . . very well written. It will prove to be equally useful to first-time investigators embarking on an adventure involving baculovirus-mediated gene expression and to those of us with considerable experience in this area who wish to have a good manual for quick reference. I would classify this book as the baculovirus equivalent of the best-selling Molecular Cloning by Sambrook, et al. and recommend it as an important primer for any lab that plans to become or is currently involved in this type of work." --Cell |k No