Attempts to express the A1-GMCSF immunotoxin in the baculovirus expression vector system

Abstract

Immunotoxins are fusion proteins consisting of two elements, a targeting and a toxin moiety, and are designed for specific elimination of tumor cells. Previously we expressed a recombinant fusion protein consisting of the toxic fragment of Shiga toxin (A1) and GMCSF (A1-GMCSF) in Escherichia coli, and evaluated its cytotoxic properties in acute myeloid leukemia and colon carcinoma cell lines. In view of the specific cytotoxic effects of this immunotoxin, further detailed in-vitro and preclinical studies were undertaken. Large amounts of the recombinant protein of high purity and free of unwanted side products, such as lipopolysaccharides (LPS), were required. Since GMCSF is of mammalian origin and it requires proper disulfide bond formation, we intended to use the baculovirus expression vector system (BEVS) for the expression of the recombinant fusion protein. However, despite previous reports on the expression of several other immunotoxins by this system, the A1 derived fusion proteins revealed an inhibitory effect on baculoviral particle formation and even caused cell death in insect cells. This observation was further pursued and confirmed by the use of other baculoviral specific promoters. The salient features of this finding are described below.