Baculovirus Expression Vectors A Laboratory Manual

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.

AcknowledgmentsHow To Use This ManualPART I: AN OVERVIEW OF BACULOVIRUSES1. Virus Structure and the Infection Process2. Gene Organization, Regulation, and Function3. Virus-Host Interactions4. Summary of Baculovirus Features Relevant to Expression FactorsPART II: CHOOSING A TRANSFER PLASMID AND PARENT VIRUS5. Choice of Virus and Host Species6. Choice of Transfer Plasmid7. Available Transfer Plasmids8. Choosing a Parent Virus for Use in Vector Construction9. Optimizing Expression: Tailoring the Heterologous Gene to the Transfer Plasmid and the Baculovirus Expression SystemPART III: METHODS FOR VECTOR CONSTRUCTION AND GENE EXPRESSION10. Safety Considerations11. Insect Cell Culture12. Virus Methods13. Contransfection and Recombinant Virus Identification14. Characterizing Recombinant Gene Expression15. Post-Translational ModificationPART IV: METHODS FOR SCALE-UP OF PROTEIN PRODUCTION AND USE OF INSECT LARVAE16. David L. Clemm: Scale-Up of Protein Production in a Stirred Bioreactor17. Cheryl Isaac Murphy: Scale-Up of Protein Production in an Airlift Fermenter18. Insect Rearing and InfectionAppendix 1: Nucleotide Sequence of AcMNPV Polyhedrin RegionAppendix 2: Nucleotide Sequence of AcMNPV p10 RegionAppendix 3: List of Genes Expressed in the Baculovirus Expression SystemAppendix 4: SuppliersAppendix 5: Stock SolutionsAppendix 6: Excel Spreadsheet for TCID50 CalculationReferencesIndex