title: A simple plasmid-based transient gene expression method using High Five cells.
abstract: The High Five (H5) cell line, derived from the lepidopteran Trichoplusia ni, is one of the major insect cell hosts for the production of recombinant proteins using the baculovirus expression vector system (BEVS). Here, we describe a simple polyethylenimine (PEI)-based transient gene expression (TGE) process for the rapid production of recombinant proteins from suspension-adapted H5 cells. The method was optimized using two model proteins, enhanced green fluorescent protein (EGFP) and human tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc). After screening several promoter and enhancer combinations for high levels of TNFR:Fc production, an expression vector containing the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer was selected. Cells were transfected at a density of 2×10(6) cells/mL by direct addition of DNA and PEI. Under optimized conditions, a 90% transfection efficiency (percentage of EGFP-positive cells) was obtained. In addition, we observed volumetric TNFR-Fc yields over 150μg/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells.
authors.affiliation: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Xiao
authors.initials: X
authors.lastname: Shen
authors.affiliation: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Ana K
authors.initials: AK
authors.lastname: Pitol
authors.affiliation: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Virginie
authors.initials: V
authors.lastname: Bachmann
authors.affiliation: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland; Protein Expression Core Facility, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: David L
authors.initials: DL
authors.lastname: Hacker
authors.affiliation: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Lucia
authors.initials: L
authors.lastname: Baldi
authors.affiliation: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland. Electronic address: florian.wurm@epfl.ch.
authors.forename: Florian M
authors.initials: FM
authors.lastname: Wurm
mesh_terms: D000818:Animals; D002452:Cell Count; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007915:Lepidoptera / Q000166:cytology*; D010957:Plasmids / Q000378:metabolism*; D011094:Polyethyleneimine / Q000737:chemistry; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D013535:Suspensions; D014162:Transfection / Q000379:methods*; D019076:Transgenes
chemical_list: D013535:Suspensions; D011094:Polyethyleneimine; D004247:DNA
keywords: Expression vector; High Five cells; Polyethyleneimine; Suspension culture; Transient gene expression