The Baculovirus Expression System : A Laboratory Guide

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 purifi...

As a result of recent major advances in molecular biological techniques, the use of baculovirus expression systems is now widespread and publications reviewing the techniques are numerous. The aim of this practical manual is to give first-time users the relevant background information and detailed laboratory methods. Methods and protocols are provided and more detailed applications are highlighted for the benefit of the more experienced user. Emphasis is placed on practical applications, interpretation of results and potential problems and pitfalls.