title: Vaccinia virus-based expression of gp120 and EGFP: survey of mammalian host cell lines.
abstract: Production of recombinant proteins with the vaccinia virus expression system in five mammalian cell lines (HeLa, BS-C-1, Vero, MRC-5, and 293) was investigated for protein yield and proper posttranslational modifications. Regulatory acceptance of the host cell line was taken into consideration, where Vero, MRC-5, and 293 were considered more acceptable to the regulatory authorities. Relevant process knowledge for ease of scale-up with the particular cell type was also considered. Two proteins were expressed, enhanced green fluorescent protein (EGFP) in the cytoplasm and gp120, an HIV envelope coat protein that is secreted into the culture medium. HeLa cells produced the most EGFP at 17.2 microg/well with BS-C-1 and 293 following. BS-C-1 produced the most gp120 at 28.2 microg/mL with 293 and Vero following. Therefore, of the three most appropriate cell lines (Vero, MRC-5, and 293) for production processes, the best results were obtained with 293 cells. Although MRC-5 had a very high productivity on a per cell basis, the low cell density and slow growth rate made the overall production insufficient. Because gp120 contained a significant amount of posttranslational modification, this protein, produced by the different cell lines, was further analyzed by PNGase digestion suggesting N-linked glycosylation modifications in all cell lines tested. On the basis of these results and overall process considerations, 293 cells are recommended for further production process optimization in a serum-free suspension system.
authors.affiliation: Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bldg 14A Rm 173, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Bleckwenn
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D012680:Sensitivity and Specificity; D013997:Time Factors; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D015699:HIV Envelope Protein gp120; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins