Continuous beta-galactosidase production in insect cells with a p10 gene based baculovirus vector in a two-stage bioreactor system.
Continuous production of polyhedra or baculovirus-expressed proteins in insect cell cultures is limited to about 4 weeks. The decrease in production has been ascribed to the interference of defective deletion mutants with wild-type baculoviruses. The deleted genome sequences include the polyhedrin gene (or the heterologous gene of interest); in the remaining part, the major late p10 gene is always maintained. In the present study, the productivity of a recombinant baculovirus with the lacZ gene from Escherichia coli cloned downstream of the p10 promoter at the p10 locus was investigated. It was hypothesized that this p10 promoter driven gene is preserved over a longer period of time in a continuously operated two-stage bioreactor system than foreign genes behind the polyhedrin promoter at the polyhedrin locus. In two separate runs, beta-galactosidase production with the p10-lacZ recombinant reached quasi-steady-state levels of 30 and 60 units/cm3. Polyhedron production was about 3 x 10(6) and 6 x 10(6) polyhedra/cm3, respectively. However, both polyhedron and beta-galactosidase production decreased after about 30 days of relatively constant production. In the infection reactor, deletion mutants of the virus, which contained both the polyhedrin and lacZ gene, were predominant. Therefore, the presence of the polyhedrin or p10 gene alone in deletion mutants is not sufficient for prolonged expression; other genes involved in major late gene expression and not present in the deleted virus are probably necessary.
van Lier
F L
FL
Department of Food Science, Wageningen Agricultural University, The Netherlands.
van Duijnhoven
G C
GC
de Vaan
M M
MM
Vlak
J M
JM
Tramper
J
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D005822:Genetic Vectors; D007763:Lac Operon; D009036:Moths; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D001616:beta-Galactosidase / Q000096:biosynthesis*
D011994:Recombinant Proteins; D001616:beta-Galactosidase