title: Virus-free transient protein production in Sf9 cells.
abstract: A method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed with the gene of interest being expressed from a plasmid carrying the homologous region 5 enhancer (hr5) and the immediate early 1 (ie1) promoter from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Under the optimal conditions described in the study, cells were transfected at a density of 30×10⁶ cells/mL with 0.9 μg DNA and 1.35 μg of linear 25 kD polyethylenimine (PEI) per million cells. Following transfection, the culture was diluted to 4×10⁶ cells/mL for the protein production phase. The volumetric yield of tumor necrosis factor receptor (ectodomain) fused to an Fc domain (TNFR-Fc) was about 100 μg/mL for cultures at volumes up to 300 mL. As expected, the molecular weight of the dimeric TNFR-Fc produced from Sf9 cells was about 6 kDa less than that produced from a recombinant Chinese hamster ovary (CHO) cell line due to differences in glycosylation between the two hosts. Transient transfection provides an alternative to the baculovirus expression vector system (BEVS) for the rapid production of recombinant proteins from Sf9 cells.
authors: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors: Xiao
authors: X
authors: Shen
authors: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors: David L
authors: DL
authors: Hacker
authors: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors: Lucia
authors: L
authors: Baldi
authors: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland. Electronic address: florian.wurm@epfl.ch.
authors: Florian M
authors: FM
authors: Wurm
mesh_terms: D000818:Animals; D016466:CHO Cells; D002452:Cell Count; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors / Q000235:genetics; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011094:Polyethyleneimine; D011401:Promoter Regions, Genetic / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061987:Sf9 Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D011094:Polyethyleneimine
keywords: Polyethylenimine; Recombinant protein; Sf9 cells; Suspension culture; Transient gene expression