Preventing proteolytic artifacts in the baculovirus expression system.

The baculovirus expression system has become established as a popular method for obtaining high levels of recombinant protein synthesis in a eukaryotic environment (5). In a number of cases, however, researchers have reported decreased product quality and yield resulting from proteolytic activity late in the course of infection (2,3,8,12). The enzyme most likely to cause this degradation is V-CATH, a cysteine protease encoded by Autographa californica M nucleopolyhedrovirus (AcMNPV) (9), the parent virus of the most common baculovirus expression vectors. V-CATH, named for its homology to the lysosomal enzyme cathepsin L, is a late viral gene product that has been shown to play an essential role in the baculovirus-induced liquefaction of the insect host (10). While much attention has been focused on attenuating VCATH activity in the later stages of infection, we have found that this protease can present problems earlier in the infection process as well. Specifically, the V-CATH proenzyme (proV-CATH) appears to be activated by the addition of sample buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol [final concentration]) during the preparation of samples for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The newly activated VCATH can then digest other proteins, yielding degradation products that are artifacts of the sample preparation. This proteolysis could be misinterpreted as an indication that recombinant protein expression is poor or that cleavage of a protein of interest is occurring in the infected cell culture, even though the protein might actually be present at high levels and otherwise very stable. As a result, time and resources could mistakenly be diverted toward attempts to solve nonexistent problems or toward exploring spurious findings. Thus, the potential for these artifacts should be of concern both to those who use the baculovirus expression system and to those who study baculovirus biology. Chemically induced activation of VCATH can be prevented by adding the protease inhibitor trans-epoxysuccinylL-leucylamido-(4-guanidino)butane (E64; Boehringer Mannheim, Indianapolis, IN, USA) to cell lysis buffers or to sample buffer. E-64 is especially wellsuited for this application because of its rapid, irreversible inhibition of cysteine proteases (1) and its stability toward reducing agents, such as those commonly found in SDS-PAGE sample buffer. We maintained cultures of Sf21 insect cells in suspension in Vaughn’s