Baculovirus and Insect Cell Expression Protocols

Robert L. Harrison, Donald L. Jarvis, Harrison, Robert L., Jarvis, Donald L.

The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, the yields of extracellular and membrane-bound proteins obtained with this system are often very low, possibly due to the adverse effects of baculovirus infection on the host insect cell secretory pathway. An alternative approach to producing poorly expressed proteins is to transform lepidopteran insect cells with the gene of interest under the control of promoters that are constitutively active in uninfected cells, thereby making cell lines that continuously express recombinant protein. This chapter provides an overview of the methods and considerations for making stably transformed lepidopteran insect cells. Techniques for the insertion of genes into continuous expression vectors, transfection of cells, and the selection and isolation of stably transformed Sf-9 clones by either colony formation or end-point dilution are described in detail.