APPEARANCE OF PROTEASE ACTIVITIES COINCIDES WITH P10 AND POLYHEDRIN-DRIVEN PROTEIN-PRODUCTION IN THE BACULOVIRUS EXPRESSION SYSTEM - EFFECTS ON YIELD

A study of proteolysis effects on recombinant protein yield was comple ted using the insect cell (Sf-9)-baculovirus (AcNPV) expression system . Activities of protease and beta-galactosidase (beta-gal), a marker h eterologous protein, were assayed at various multiplicities of infecti on (MOI = 1, 5, and 20) on a time course postinfection. Also, several protein-substrate gel electrophoresis assays were run using gelatin, b eta-gal, and bovine serum albumin (BSA) in the gel matrix, to determin e the protein specificity of the proteases. The most abundant protease activity (cysteine), found at 49 kDa, degraded all three substrates, pre-and post-infection. Two other protease activities (40 and 36 kDa) appeared on polyacrylamide gel electrophoresis (PAGE) gels after 72 hp i (hours postinfection). In addition, the culture with the highest MOI had the highest beta-gal activity until 72 hpi, when the activity dra matically decreased coincidentally with a 2.5-fold increase in proteas e activity. This result and the electrophoresis evidence that the prot ease is specific to beta-gal, indicate that there is a negative correl ation between protease activity and recombinant protein yield, These r esults guide efforts to control product-degrading proteolysis in insec t cell-baculovirus expressions systems by harvest timing and the addit ion of protease inhibitors.