In vitro and in vivo RNA interference mediated suppression of Tn‐caspase‐1 for improved recombinant protein production in High Five^TMcell culture with the baculovirus expression vector system

Abstract

While traditional metabolic engineering generally relies on the augmentation of specific genes and pathways in order to increase the yield of target proteins, the advent of RNA interference (RNAi) as a biological tool has given metabolic engineers another tool capable of rationally altering the host cell's biological landscape in order to achieve a specific goal. Given its broad applicability and potent specificity, RNAi has the ability to suppress genes whose function is contrary to the desired phenotype. In this study, RNAi has been used to increase recombinant protein production in a Trichoplusia ni derived cell line (BTI-TN-5B1-4-High Five) using the Baculovirus Expression Vector System. The specific target investigated is Tn-caspase-1, a protease involved in apoptosis that is likely the principal effector caspase present in T. ni cells. Experiments were first conducted using in vitro synthesized dsRNA to verify silencing of Tn-capase-1 and increased protein production as a result. Subsequent experiments were conducted using a cell line stably expressing in vivo RNAi in the form of an inverted repeat that results in a hairpin upon transcription. Using this construct, Tn-caspase-1 transcript levels were decreased by 50% and caspase enzymatic activity was decreased by 90%. This cell line, designated dsTncasp-2, demonstrates superior viability under low nutrient culture conditions and resulted in as much as two times the protein yield when compared to standard High Five cells.

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

“…Although luciferase expression did not correlate with accumulated SEAP expression, it was apparent that both extracellular and intracellular recombinant protein production in Sf -caspase-1-repressed stable cells was higher than for parental cells (Figures 4 and 5). Thus, these data indicate that Sf -caspase-1-repressed stable cells express a higher amount of recombinant protein in BEVS, consistent with previous studies in S. frugiperda cells by our group [25] and in T. ni cells by Bentley’s group [24,34]. Hence, we can suggest that the apoptotic repressed insect cells have greater recombinant protein production when infected with recombinant baculovirus, providing an effective production tool for BEVS.…”

“…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. Therefore, in this present study, we use RNAi-mediated Sf -caspase-1-repressed stable cells to clarify how resistance to apoptosis could enhance both intracellular (firefly luciferase) and extracellular (secreted alkaline phosphatase [SEAP]) recombination protein production by BEVS.…”

“…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.…”

Baculovirus-mediated Gene Delivery and RNAi Applications

“…AcMNPV encodes miRNAs that lead to a reduction of BVs and accelerated formation of ODVs [ 243 ]. The RNAi has been shown to persists for four to eight days in baculovirus-infected as well as uninfected Sf9 cells [ 232 ].The RNAi-approach has been also used to increase recombinant protein production in a Trichoplusia ni derived cell line (BTI-TN-5B1-4-High Five) [ 244 ].…”

Special Issue: Gene Therapy with Emphasis on RNA Interference

“…In this context, Makkonen and co-authors review applications of baculovirus-based RNAi. For example, prevention of AcMNPV infection was obtained by RNAi delivery [ 7 ] and recombinant protein production was increased in Trichoplusia ni by an RNAi-approach [ 8 ]. Furthermore, as an example of an RNAi-based application in mammalian cells, baculovirus U6-driven short hairpin RNA (shRNA) efficiently decreased lamin A/C mRNA and protein levels in liver cell lines and in primate human hepatic stellate cells [ 9 ].…”