Sf‐Caspase‐1‐repressed stable cells: resistance to apoptosis and augmentation of recombinant protein production

Abstract

Sf-Caspase-1 [Spodoptera frugiperda (fall armyworm) caspase-1] is the most studied effector caspase of Lepidoptera and its activation may lead cells to apoptosis (programmed cell death) when under UV irradiation or baculovirus infection. In the present study, we repressed the expression of Sf-caspase-1 in Sf9 (S. frugiperda 9) cells using constitutive RNA interference, and evaluated the effects of stress responses and the production of proteins in recombinant baculovirus-infected cells. The Sf-caspase-1-repressed stable cells, Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2, showed a significant increase in resistance to UV- and baculovirus-induced apoptosis. These cells produced higher levels of both intracellular (luciferase) and extracellular [SEAP (secreted alkaline phosphatase)] recombinant proteins than the parental cells when infected with recombinant baculovirus. Thus Sf-caspase-1-repressed stable cells have a greater ability to adapt to various culture conditions, and also to provide the benefits of high-level protein production.

Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

“…These results demonstrated that Sf -caspase-1 mRNA was successfully suppressed by Sf -caspase-1 dsRNA in both Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2 cells. In our previous study, we tested the expression of P35, a substrate inhibitor of Sf -caspase-1, in Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2 cells, and found that the level of P35 in pIBdsCasp transfected cells was evidently higher than that found in normal cells [25]. Thus, we suggested that Sf -caspase-1 is suppressed in these Sf -caspase-1-repressed cells.…”

“…Stable cells (Sf9/pIB, Sf9/pIBdsCasp-1, and Sf9/pIBdsCasp-2 cells) were established by transfection of the control vector pIB or vector pIBdsCasp (Figure 2), and subsequently selected by BSD [25]. After more than 20 passages, stable cell lines were analyzed by genomic DNA PCR and RT-PCR to examine the inverted-repeat DNA sequence of Sf -caspase-1 and endogenous expressed Sf -caspase-1 mRNA quantity.…”

“…Few studies have examined the potential of RNA inference (RNAi) to increase protein production in the BEVS [24,25], however, several studies have demonstrated the efficiency of this approach in both insect cells and larvae [26,27]. In our previous studies, we used DNA vector-based approaches with endogenously expressed double-stranded RNA (dsRNA) to silence its target gene, Sf -caspase-1, in Sf9 cells.…”

“…In our previous studies, we used DNA vector-based approaches with endogenously expressed double-stranded RNA (dsRNA) to silence its target gene, Sf -caspase-1, in Sf9 cells. Therefore, the Sf -caspase-1 mRNA level and plasmid copy number in the Sf -caspase-1-repressed stable cells were examined [25]. In addition, Sf -caspase-1-repressed stable cells exhibited resistance to apoptosis and enhancement of recombinant protein production [25,28].…”

“…Therefore, the Sf -caspase-1 mRNA level and plasmid copy number in the Sf -caspase-1-repressed stable cells were examined [25]. In addition, Sf -caspase-1-repressed stable cells exhibited resistance to apoptosis and enhancement of recombinant protein production [25,28]. These results were consistent with later findings in T. ni cells [24]; however, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells was not determined.…”