title: Production of rotavirus core-like particles in Sf9 cells using recombinase-mediated cassette exchange.
abstract: A flexible Sf9 insect cell line was recently developed leveraging the recombinase-mediated cassette exchange (RMCE) technology, which competes with the popular baculovirus expression vector system (BEVS) in terms of speed to produce new proteins. Herein, the ability of this cell platform to produce complex proteins, such as rotavirus core-like particles, was evaluated. A gene construct coding for a VP2-GFP fusion protein was targeted to a pre-characterized high recombination efficiency locus flanked by flipase (Flp) recognition target sites and, after three weeks in selection, an isogenic cell population was obtained. Despite the lower cell specific productivities with respect to those obtained by baculovirus infection, the titers of VP2-GFP reached in shake flask batch cultures were comparable as a result of higher cell densities. To further improve the VP2-GFP levels from stable expression, analysis of exhausted medium was undertaken to design feeding strategies enabling higher cell densities as well as increased culture duration. The implementation of the best strategy allowed reaching 20 million cells per ml in bioreactor cultures; the integrity of the rotavirus core-like particles could be confirmed by electron microscopy. Overall, we show that this Sf9-Flp cell platform represents a valuable alternative to the BEVS for producing complex recombinant proteins, such as rotavirus core-like particles.
authors: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors: Fabiana
authors: F
authors: Fernandes
authors: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors: Mafalda M
authors: MM
authors: Dias
authors: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors: João
authors: J
authors: Vidigal
authors: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors: Marcos F Q
authors: MF
authors: Sousa
authors: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors: Marco
authors: M
authors: Patrone
authors: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors: Ana P
authors: AP
authors: Teixeira
authors: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. Electronic address: marques@itqb.unl.pt.
authors: Paula M
authors: PM
authors: Alves
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019149:Bioreactors; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D045522:Recombinases / Q000235:genetics*; D012401:Rotavirus / Q000235:genetics*; D061987:Sf9 Cells; D018411:Spodoptera; D014771:Virion / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D045522:Recombinases; D049452:Green Fluorescent Proteins
keywords: BEVS; Flp-mediated site-specific integration; Rational process optimization; Rotavirus core-like particles; Virus-free insect cell expression