Preparing Antigens Using a Baculovirus Expression System.

Abstract

Baculovirus-produced recombinant proteins circumvent many of the issues that limit bacterial protein production. Baculoviruses contain double-stranded, circular, supercoiled DNA in a rod-shaped capsid. The viral life cycle includes three major phases: (1) early (or virus synthesis) phase (0.5-6 h after infection), which includes viral attachment, uncoating early viral gene expression, and shutting off host gene expression; (2) late phase (6-12 h after infection), which includes viral replication and formation of budded virus containing host plasma membrane and glycoprotein (gp)64, which is required for virus entry; and (3) very late phase (24-96 h after infection), which includes formation of polyhedral inclusion bodies and cell lysis. Large proteins can thus be produced readily in abundant quantities. Furthermore, posttranslational modifications of the protein similar to those of mammals (such as phosphorylation, disulfide-bond formation, and even glycosylation) will occur; indeed, because typical baculovirus constructs produce secreted proteins, most products will be glycosylated. Baculovirus expression systems are safe, because baculoviruses are nonpathogenic to mammals and plants, and insect cells can be grown easily in suspension culture, which makes protein production easy to scale up. Counterbalancing this approach, however, is the need to perform a two-step process: first cloning the gene into the recombination donor vector and then generating the viral construct. At that point, the virus can be used as a mixture or plaque-purified. Insect cells are then infected with the virus, and the proteins are produced.