Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method.
Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions.
Department of Pharmacy, University of Patras, GR 26500, Patras, Greece.
Athanasios
A
Niarchos
Department of Pharmacy, University of Patras, GR 26500, Patras, Greece.
George
G
Lagoumintzis
Department of Pharmacy, University of Patras, GR 26500, Patras, Greece.
Konstantinos
K
Poulas
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics*; D018929:Cell Culture Techniques; D002460:Cell Line; D005786:Gene Expression Regulation / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014775:Virus Activation / Q000235:genetics
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins