Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors

In insect cell-baculovirus expression systems for recombinant protein produ ction, it is sometimes necessary to supplement cultures with protease inhib itors to protect recombinant proteins against proteolysis. To date, however , there is no information available concerning protease activities in inhib itor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-i nfected Sf-9 insect cells which were supplemented with inhibitors against c arboxyl and cysteine proteases produced during culture. Prior to the supple mentation culture, the cell toxicity of several protease inhibitors was det ermined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cy statin, leupeptin, and antipain (cysteine protease inhibitors) tested in th is study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit th e respective intracellular protease activities. A virus-infected culture wi th a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protei n biosynthesis was reduced by 5-30%. However, a significant reduction in ca rboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrat es a reduction in extracellular and intracellular protease activities in pr otease inhibitor-supplemented cultures of virus-infected insect cells.