Experiments undertaken with commercially available recombinantly produced human immunodeficiency virus Type 1 (HIV-1) gp120 demonstrated that the resuspended lyophilized protein, a product of the baculovirus expression system, had intrinsic nuclease activity. This nuclease activity was distinguishable from the moleculargrade bovine serum albumin that it was constituted in. The activity was thermolabile in that if the preparation was heated to 100°C for 10 min, the activity was abolished, although this did not happen when it was stored at -20°C. The nuclease activity was also
INTRODUCTIONThe successful use of the insect cell/baculovirus expression strategy in the large-scale production of recombinant proteins of mammalian, insect, plant and bacterial origin has revolutionized biology, biotechnology and chemistry. This highly efficient expression system has been well characterized (6,9,11), and its ubiquitous utilization and exploitation is testament to the many factors intrinsic to the system that contribute to its popularity. These include high protein expression levels, ease and speed of genetic engineering, accommodation of large DNA inserts, post-translational protein processing (e.g., glycosylation) similar to mammalian cells and ease of insect cell growth in suspension (11).Despite years of intensive investigation and speculation concerning the specific virological and immunological mechanisms of cell death involved in human immunodeficiency virus Type 1 (HIV-1) infection (13), certain of these still remain elusive. Nevertheless, there is mounting and conclusive evidence that the process of apoptosis is at least contributory to T-cell depletion (1,10,19,20).Since the gp120 protein has been incontrovertibly linked to apoptotic cell death mechanisms in HIV-1 disease pathogenesis, and because there are dangers inherent in the purification of gp120 from cultures infected with the virus and from blood derived from HIV + donors, gp120 is often produced in the insect/baculovirus system for subsequent use in research and commercial enterprises.In this study, which was aimed at the evaluation of putative apoptosis-blocking strategies in cultures of peripheral blood mononuclear cells (PBMC) exposed to cross-linked gp120/anti-gp120 complexes, we noted contaminating endonuclease activity in culture supernatants containing recombinant gp120 protein that had been produced in the baculovirus expression system. While not disputing the role of apoptosis in Tcell depletion in AIDS, this report is aimed at drawing attention to the baculovirus-associated endonuclease activity, which may confound the interpretation of studies addressing this phenomenon in vitro.
MATERIALS AND METHODS
Use of Special ReagentsBaculovirus-produced recombinant gp120 IIIB and IgGpurified rabbit anti-gp120 antibodies were obtained from Intracel (London, England, UK or Cambridge, MA, USA) and were reconstituted in sterile double-distilled water. Molecular biology-grade bovine serum albumin (BSA), which contained no deoxyribonucleases, ribonuc...
