title: Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.
abstract: The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.
authors.affiliation: Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Bleckmann
authors.affiliation: Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Margitta
authors.initials: M
authors.lastname: Schürig
authors.affiliation: Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Fang-Fang
authors.initials: FF
authors.lastname: Chen
authors.affiliation: Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Zen-Zen
authors.initials: ZZ
authors.lastname: Yen
authors.affiliation: Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Lindemann
authors.affiliation: Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Meyer
authors.affiliation: Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Spehr
authors.affiliation: Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Joop
authors.initials: J
authors.lastname: van den Heuvel
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D007313:Insecta / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D061987:Sf9 Cells; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection / Q000379:methods
chemical_list: D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins