RNA interference mediated suppression of Tn-caspase-1 as a means of investigating apoptosis and improving recombinant protein production in Trichoplusia ni cells.

摘要

The baculovirus expression system has proven to be a robust and versatile system for recombinant protein production in insect cells. A wide range of promoters is available for the facile expression of transgenes, and yields of up to 50% of total protein have been reported. However, in many cases production is decreased as a result of proteases and host cell apoptosis. To combat this problem, RNA interference (RNAi) has been used as a metabolic engineering tool to knockdown host genes responsible for decreasing the yield of recombinant protein. A novel caspase (Tn caspase-1) derived from Trichoplusia ni cells has been identified and characterized. Through modulation of caspase levels via either RNAi or through interaction with baculovirus protein p35, the overall level of apoptosis present in cell culture has been decreased. In addition, the use of in vitro RNAi targeted against Tn caspase-1 has increased the production of recombinant green fluorescent protein. To further study the effect of suppressing Tn caspase-1, a stable cell line producing in vivo RNAi was developed, resulting in a nearly 90% decrease in caspase enzymatic activity. This suppression was able to improve culture viability under adverse conditions and increase recombinant protein production levels up to two-fold that of standard cells.