Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter.
S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained approximately 1.5-3 x 10(7)cells/mL after 3-4 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 microg/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 microg/10(7) cells at days 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 microg/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function.
Ventini
Daniella Cristina
DC
Laboratório de Imunologia Viral, Instituto Butantan, Avenida Vital Brasil 1500, São Paulo 05503-900, SP, Brazil.
Astray
Renato Mancini
RM
Lemos
Marcos Alexandre Nobre
MA
Jorge
Soraia Attie Calil
SA
Riquelme
Camilo Calderón
CC
Suazo
Claudio Alberto Torres
CA
Tonso
Aldo
A
Pereira
Carlos Augusto
CA
D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques; D048708:Cell Growth Processes / Q000502:physiology; D002460:Cell Line; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011820:Rabies virus / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D006023:Glycoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D010100:Oxygen