Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in high five (TM) cells

The insect cell line BTI-TN-5B1-4 (High Five(TM)) is frequently used to exp ress recombinant proteins ill large amounts using the baculovirus expressio n system. However extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant prote ins migrate in SDS-PACE in agreement,with poly-ubiquitinated forms of the p rotein, suggesting a ubiquitin/proteasome degradation pathway Here, we desc ribe a systematic study unraveling the effect of adding proteasome inhibito rs or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibit ors were added to the lysis buffer to establish the most efficient way to i nhibit proteolytic activity after lysis of baculovirus-infected cells expre ssing recombinant proteins. We conclude that a combination of adding protea se inhibitors to the growth medium and to the lysis buffer minimizes the pr oteolytic activity in High Five cells. The most efficient protease inhibito rs were E-64 in the growth medium together with Leupeptin in the lysis buff er at concentrations higher than with available cocktails of inhibitors. Th e optimal treatment of High Five cells is different from the optimal treatm ent of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein .