Improved Protein Manufacturing in Insect Expression Systems

The central theme of the proposed research is improvement of the quality of product proteins manufactured in insect larvae. The approach is twofold: create the most favorable conditions for the expressed protein to mature, and create the least favorable conditions for proteolytic breakdown in the insect and through downstream processing. A number of molecular biological adaptations to baculovirus expression vectors are proposed to increase the proportion of correctly folded, matured, and biologically active protein expressed. The other objectives all deal with maintaining the quality of the produced product, in the insect as well as during downstream processing. We will undertake a biochemical characterization of the proteolytic activity in infected insects, and use fluorescent reporter genes to monitor protease load. We will also characterize the type and quantity of proteolytic activity through the steps of protein separation and purification. Protease degradation in the insect will be addressed through the use of protease inhibitors and interfering RNA.