RESUMEN
Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.
Asunto(s)
Proteínas Recombinantes/biosíntesis , Transfección/métodos , Reactores Biológicos , Western Blotting , Línea Celular , Medios de Cultivo , Proteínas de Unión al ADN , Electroforesis en Gel de Poliacrilamida , Antígenos Nucleares del Virus de Epstein-Barr/genética , Escherichia coli/genética , Expresión Génica , Genes Reporteros , Marcadores Genéticos , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Inmunoglobulina G/genética , Proteínas Luminiscentes , Microscopía Confocal , Plásmidos , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT3 , Proteínas Recombinantes/genética , Factores de TiempoRESUMEN
Human serum albumin (HSA) and human gamma globulin (HGG) in serum and uterine fluid of nonpregnant rabbits at various times after an i.v. injection (100 mg/kg) were measured by a radial immunodiffusion test using specific antisera. The HSA concentration in uterine fluid rose to a peak at 12 hr when it was 11% of the serum concentration and then declined, whereas HGG reached a peak at 18 hr (3.2% of serum level) and decreased thereafter. The HSA passed 2 1/2 times faster than HGG, but both proteins equilibrated with uterine fluid in about 12-18 hr. Steady state levels of HSA and HGG indicated that uterine fluid: serum ratios were 1:10 and 1:20, respectively. Similar ratios were found for total protein and rabbit serum albumin (1:10) and rabbit gamma globulin (1:20). Therefore, except when there is a local immune response, the uterine lumen contains only about 5% of the serum antibody concentration. Available data in the mouse, rat and dog also indicate disparity between serum and uterine fluid protein levels.