In vitro and in vivo RNA interference mediated suppression of Tn-caspase-1 for improved recombinant protein production in High Five cell culture with the baculovirus expression vector system.
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.
Hebert
Colin G
CG
Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Science Building, College Park, Maryland 20742, USA.
Valdes
James J
JJ
Bentley
William E
WE
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D020170:Caspase 1 / Q000235:genetics; D061945:Caspase Inhibitors; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002470:Cell Survival; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D007915:Lepidoptera; D034622:RNA Interference; D011994:Recombinant Proteins / Q000096:biosynthesis*
D061945:Caspase Inhibitors; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D020170:Caspase 1