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.

Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells

“…To establish the calibration curve centrifuged cells were resuspended in media and diluted to respective concentrations. With the recommended shaking speed of 700 rpm for high insect cell concentrations 11 , we observed a high tendency of cells to settle and build up a layer on the bottom of the well. This led to a saturation of the scattered light signal, even at low cell densities, and consequently false cell count estimates.…”

“…This high-throughout (HTP) µ-bioreactor system enables online monitoring of cell density, fluorescence, DO level, and pH in a continuously shaken 2.5 mL volume and has already been described as being well-suited for Sf9 insect cells 11 . Continuous scattered light measurement offers the possibility of obtaining real-time information on cell growth during cultivation.…”

Capsid Proteins are Necessary for Replication of a Parvovirus

“…We expected that viral genes moved outside of the genome would experience a relative decrease in gene dosage, as the viral genome is replicated. In our design, we had sought to compensate this by using one of the strongest heterologous insect promoter (24), the immediate early 2 promoter from the multicapsid Nuclear Polyedrosis Virus of Orgyia pseudotsugata (POpIE2), to drive ns expression. As insufficient expression of NS functions would effect inefficient replication rather than its complete shutdown, we conjectured that the observed defect could rather stem from an ineffective expression pattern linked to the absence of appropriate regulatory signals in the heterologous promoter sequence.…”

Identifying parameters to improve the reproducibility of transient gene expression in High Five cells

“…In order to improve the throughput and sensitivity of detection of successful expression constructs, fast screening strategies using TGE combined with transactivation [29, 30] or the SplitGFP technology [31] have been established. These methods avoid the need to generate high amounts of different recombinant baculoviruses.…”