title: Production of HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 expression system.
abstract: The HeLa cell-vaccinia virus system is an attractive method for producing recombinant mammalian proteins with proper post-translation modifications. This approach is especially important for the production of HIV-1 envelope glycoprotein, gp120, since more than half of its total mass is due to carbohydrates. A recombinant vaccinia virus/T7 RNA polymerase expression system was developed to express and produce large amounts of gp120 tagged with six histidine residues. In this system, the expressed T7 RNA polymerase from one virus drives the transcription of the gp120 encoded in the second virus. During the process development phase, the following parameters were studied: infection time, infection duration, multiplicity of infection, ratio of the two viruses, medium composition, and medium replacement strategy during the infection phase. The chosen production method was based on using the packed-bed bioreactor. The HeLa cells were immobilized on fibrous disks (Fibra-Cel) packed in an internal basket positioned in a vertically mixed bioreactor (Celligen Plus), and 25 g of carriers were packed in a 1.6-L (working volume) reactor. The process included a growth stage followed by a production stage. In the growth stage, the bed was perfused with a serum-containing medium, allowing the cells to grow to saturation, and in the production stage, done using serum-free medium, the cells were infected with the two recombinant viruses. The expressed protein was secreted, collected from the culture fluid, and purified. The specific production was found to be between 2 and 3 microg of protein/10(6) cells, and the volumetric production was around 10 mg/50 g carriers.
authors.affiliation: Biotechnology Unit, LCDB, NIDDK, and Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda, Maryland 20892, USA.
authors.forename: Y C
authors.initials: YC
authors.lastname: Hu
authors.forename: J
authors.initials: J
authors.lastname: Kaufman
authors.forename: M W
authors.initials: MW
authors.lastname: Cho
authors.forename: H
authors.initials: H
authors.lastname: Golding
authors.forename: J
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics*; D019149:Bioreactors; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis* / Q000235:genetics; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics*
chemical_list: D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins