The Baculovirus Expression System

1 The baculoviruses.- 1.1 Introduction.- 1.2 Isolation and host range.- 1.3 Structure and classification.- 1.4 Baculovirus replication in vivo.- 1.5 Baculovirus replication in vitro.- 1.5.1 Baculovirus gene expression and replication.- 1.5.2 Baculovirus gene promoters.- 1.6 Genetic engineering of baculovirus insecticides.- 2 The development of baculovirus expression vectors.- 2.1 Introduction and historical perspective.- 2.2 The merits of the baculovirus expression system.- 2.2.1 Advantages.- 2.2.2 Disadvantages.- 2.3 General principles for inserting foreign genes into the baculovirus genome.- 2.4 Baculovirus transfer vectors.- 2.4.1 Polyhedrin promoter-based expression vectors.- 2.4.2 p10 promoter-based transfer vectors.- 2.4.3 Multiple expression vectors.- 2.4.4 Transfer vectors utilizing other baculovirus gene promoters.- 2.5 Selection of recombinant viruses.- 2.5.1 Selection of a polyhedrin-negative phenotype.- 2.5.2 Selection of f3-galactosidase-negative viruses.- 2.5.3 Recombinant virus selection using dot-blot hybridization.- 2.5.4 Screening for a positive phenotype.- 2.5.5 Enhancing the numbers of recombinant viruses.- 3 Processing of foreign proteins synthesized using baculovirus vectors in insect cells.- 3.1 Introduction.- 3.2 Glycosylation.- 3.3 Phosphorylation, acylation and amidation.- 3.4 Proteolytic processing.- 3.5 Cellular targeting and secretion.- 3.6 Tertiary and quaternary structure formation.- 3.7 Expression of viral genes.- 3.8 Expression of bacterial and fungal genes.- 3.9 Post-transcriptional processing.- 4 Construction of transfer vectors containing the foreign gene.- 4.1 Introduction.- 4.2 Isolation of foreign gene coding sequences.- 4.2.1 Some general guidelines.- 4.2.2 Isolation of DNA fragments from agarose gels.- 4.3 Modifying the ends of DNA molecules.- 4.3.1 Mung bean nuclease.- 4.3.2 Klenow fill-in.- 4.4 Preparation of the transfer vector.- 4.5 DNA ligations.- 4.6 Transformation of bacteria.- 4.7 Screening for recombinant baculovirus transfer vectors.- 4.7.1 Colony hybridization.- 4.7.2 Rapid isolation of bacterial plasmid DNA (mini-preps).- 4.8 Analysis of recombinant transfer vectors.- 4.9 Isolation of highly purified plasmid DNA (maxi-preps).- 5 Insect cell culture media and maintenance of insect cell lines.- 5.1 Introduction.- 5.2 Cell lines.- 5.3 Culture media.- 5.4 Preparation of culture media.- 5.4.1 Preparation of TC100/FCS growth medium.- 5.4.2 Preparation of Grace’s (TNM-FH) growth medium.- 5.4.3 Preparation of 4.5 1 TC100 medium from powdered formula.- 5.4.4. Preparation of TC100 medium from individual ingredients.- 5.4.5 Specialized TC100 media.- 5.4.6 Alternative insect cell culture media.- 5.5 Glassware and disposable plasticware.- 5.5.1 Suggested cleaning regime for tissue culture glassware.- 5.6 Insect cell culture.- 5.6.1 Routine sub-culturing of Sf cell lines (monolayer cultures).- 5.6.2 Routine sub-culturing of Sf cells maintained in spinner cultures.- 5.7 A guide to Sf cell seeding densities for experimental work.- 5.8 Freezing, storage and recovery of insect cells in liquid nitrogen.- 5.8.1 Freezing and storage of cells in liquid nitrogen.- 5.8.2 Recovery of cells from liquid nitrogen.- 5.9 A guide to adapting cells to serum-free media.- 6 Propagation, titration and purification of AcMNPV in cell culture.- 6.1 Introduction.- 6.1.1 Safety considerations: general rules for working with baculoviruses.- 6.2 Infection of cells with virus for experimental work.- 6.2.1 Infection of Sf cells in monolayer culture.- 6.2.2 Infection of Sf cells in suspension culture.- 6.3 Titration of virus by plaque-assay.- 6.3.1 Standard plaque-assay.- 6.3.2 Plaque-assay of lacZ-positive viruses.- 6.4 Plaque-picking and plaque-purification.- 6.5 Amplification of virus stocks.- 6.5.1 To prepare a seed stock of virus from a plaque-pick.- 6.5.2 Preparation of an intermediate stock of virus.- 6.5.3 Preparation of a high-titre working stock of virus.- 6.6 Large-scale production of virus for the purification of virus particles.- 6.7 Purification of infectious virus DNA.- 6.8 Titration of virus by TCID50.- 7 Production and selection of recombinant virus.- 7.1 Introduction.- 7.2 Preparation of linear AcMNPV.lacZ (or AcMNPV.SC) DNA.- 7.3 Co-transfection of insect cells.- 7.3.1 Co-transfection by lipofection.- 7.3.2 Co-transfection by calcium phosphate co-precipitation.- 7.4 Separation of parental and recombinant viruses by plaque-assay.- 7.5 Plaque-purification and amplification of recombinant virus stocks.- 7.6 Amplification and detection of recombinant viruses by limiting dilution and dot-blot hybridization.- 8 Characterization of recombinant viruses.- 8.1 Introduction.- 8.2 Analysis of recombinant virus genomes.- 8.2.1 Extraction of DNA from virus-infected cells.- 8.2.2 Analysis of virus DNA with restriction endonucleases.- 8.2.3 Southern hybridization analysis of virus DNA.- 8.3 Analysis of foreign gene expression by polyacrylamide gel electophoresis, using unlabelled or radiolabelled cell proteins.- 8.3.1 Radiolabelling proteins in virus-infected insect cells.- 8.3.2 Polyacrylamide gel electrophoresis of infected cell extracts.- 8.4 Analysis of recombinant protein synthesis in insect cells using immunological techniques.- 8.4.1 Immunofluorescence.- 8.4.2 Western blot analysis of virus-infected cell proteins.- 8.4.3 Immunoprecipitation of virus-infected cell proteins.- 8.5 Analysis of post-translational processing events in insect cells.- 8.5.1 Glycosylation.- 8.5.2 Phosphorylation.- 8.5.3 Palmitylation and myristylation.- 8.6 Analysis of transcription in recombinant virus-infected cells.- 8.6.1 Extraction of RNA from insect cells.- 8.6.2 Analysis of RNA using Northern blot hybridization.- 9 Scaling up the production of recombinant protein in insect cells; laboratory bench level.- 9.1 Introduction.- 9.2 Large-scale culture of insect cells.- 9.2.1 Large-scale culture of insect cells in monolayer cultures.- 9.2.2 Large-scale culture of insect cells in suspension cultures.- 9.3 The importance of highly infectious virus stocks.- 9.4 Multiplicity of infection.- 9.5 The optimum time to harvest virus-infected cells.- 9.6 Purification of recombinant protein from infected cell cultures.- 10 Propagation of baculoviruses in insect larvae.- 10.1 Introduction.- 10.2 Rearing insects in the laboratory.- 10.3 Infection of insect larvae with polyhedra from cell culture.- 10.3.1 Preparation of virus polyhedra from infected cells in rulture.- 10.3.2 Propagating the virus in insect larvae.- 10.4 Purification of polyhedra from infected larvae.- 10.5 Bioassays of polyhedra.- 10.5.1 LD50 assays.- 10.5.2 LT50 assays.- 10.6 Purification of virus particles and DNA from polyhedra.- 10.7 Isolation of virus particles from infected larvae to establish infections in cell culture.- 10.7.1 Purification of virus particles from polyhedra for the infection of cells in culture.- 10.7.2 Purification of virus particles from haemolymph for the infection of cells in culture.- 10.8 Preparation of semi-synthetic insect diet.- 11 Trouble-shooting guide.- 11.1 Introduction.- 11.2 Insertion of foreign gene coding sequences into transfer vectors.- 11.2.1 The transfer vector.- 11.2.2 DNA sequences for insertion into transfer vectors.- 11.2.3 Ligations.- 11.3 Cell culture.- 11.3.1 Cells fail to thrive and attach to glass/plastic surfaces.- 11.3.2 Cells are contaminated with virus.- 11.3.3 Cells are contaminated with yeast, fungi or bacteria.- 11.3.4 Crystals of precipitate in the medium.- 11.4 Preparation of virus stocks and infectious DNA.- 11.4.1 Virus stocks.- 11.4.2 Infectious virus DNA purification.- 11.5 Co-transfections.- 11.6 Baculovirus plaque-assays.- 11.6.1 Condition of the cells.- 11.6.2 Plaque-assay manipulations.- 11.6.3 General problems.- 11.7 Screening for recombinant viruses.- 11.8 Instability of recombinant viruses.- 11.9 Poor yields of recombinant protein.- Appendix A list of selected suppliers.- References.