Purification of Recombinant Baculovirus by End-Point Dilution and gp64 Screening

by Brooks Hayes and Thera Mulvania, PhD
Volume 12, Issue 3 (Fall 2013)

The baculovirus expression vector system (BEVS) has emerged as a powerful tool for the production of recombinant proteins used as therapeutics, reagents, and diagnostics. In order to maximize the system’s efficiency and thereby reduce costs, optimizing production parameters is imperative. A critical factor in optimization is the production of a high-quality baculovirus stock with a high-titer, pure clonal population of recombinant virus that is stable over time. Baculovirus stocks may contain alternate varieties of infectious virus due to cross-contamination, outgrowth of non-recombinant virus, and excision of inserts attributable to some recombinant virus production technologies. Since the advent of the BEVS, the “gold standard” for production of pure baculovirus stocks has been plaque purification. Briefly, plaque purification involves infecting a monolayer of cells with dilutions of virus before applying an agarose overlay to the monolayer. After a 5–7 day incubation, isolated plaques can be picked, virus eluted from the agarose plug, and amplified. The drawbacks of plaque purification are: (1) it is time and labor-intensive; (2) the results hinge greatly on the health of the cells and the cell density at infection; (3) identification and picking of isolated plaques is challenging; and (4) the integrity of the procedure is easily compromised by virus diffusion and mass flow of virus-containing liquid beneath the agarose overlay...

Citation:
Hayes B, Mulvania T. Purification of Recombinant Baculovirus by End-Point Dilution and gp64 Screening. BioProcess J, 2013; 12(3): 20-27.
http://dx.doi.org/10.12665/J123.Hayes.

Posted online September 30, 2013.