Determination of Recombinant Baculovirus Display Viral Titer

Abstract

INTRODUCTIONThe baculovirus expression vector system has been used extensively to produce numerous proteins originating from both prokaryotic and eukaryotic sources. In addition to easy cloning techniques and abundant viral propagation, the system’s insect cell environment provides eukaryotic post-translational modification machinery. The baculovirus display vector system provides a number of advantages over prokaryotic systems, allowing the combination of genotype with phenotype, enabling presentation of foreign peptides or even complex proteins on the baculoviral envelope or capsid. Baculoviruses permit larger gene insertions, are easily propagated, and can be grown to high titers. Furthermore, surface modifications of the viral capsid enable specific targeting. This strategy can be used to enhance viral binding and entry to a wide variety of both dividing and nondividing mammalian cells, as well as to produce antibodies against the displayed antigen. In addition, the technology should enable modifications of intracellular behavior, i.e., trafficking of recombinant “nanoparticles,” a highly relevant feature for studies of targeted gene or protein delivery. Although baculovirus titer can be determined by standard methods such as classical plaque assays or end-point dilution assays, such methods often are tedious and time-consuming. The protocol described here is rapid and can be performed directly using marker genes such as green fluorescent protein or β-galactosidase regulated by baculovirus-specific promoters, or indirectly as an immunoassay with baculovirus-specific antibodies (e.g., anti-gp64).