PRODUCTION OF A RECOMBINANT IMPORTED FIRE ANT VENOM ALLERGEN, SOL-I-2, IN NATIVE AND IMMUNOREACTIVE FORM

Background: The complementary DNA encoding for the important imported fire ant venom allergen, Sol i 2, has previously been cloned. The bind ing of human IgE antibodies to Sol i 2 has been demonstrated to be con formation-dependent. Methods: A complete cDNA clone encoding the Sol i 2 protein sequence and its natural signal sequence has been produced by polymerase chain reaction. The clone was ligated into a pBluebac II I transfer vector (Invitrogen Corp., San Diego, Calif), and the recomb inant baculovirus was isolated by plaque purification. The recombinant baculovirus was grown in Sf9 and High-Five cells (Invitrogen Corp.) i n serum-free media. The recombinant Sol i 2 was isolated and character ized. Results: Recombinant (r) Sol i 2 was produced in microgram/per m illiliter amounts in Sf9 cells and at 30 mu g/ml in High-Five cells. I t was isolated by ultrafiltration and reverse-phase chromatography. Th e rSol i 2 demonstrated similar binding to natural-Sol i 2 in both a c onformation-dependent ELISA assay and in RAST with sera from patients allergic to Sol i 2. The N-terminal sequence of the rSol i 2 was ident ical to that of the natural molecule. No significant increase in bindi ng activity was found after treatment of rSol i 2 with protein disulfi de isomerase. The binding of rSol i 2 to a conformation-dependent mono clonal antibody was lost by heating in sodium dodecylsulfate and reduc tion. Conclusions: A recombinant Sol i 2 protein was produced at high yield in a baculovirus expression system by using serum-free medium wi th a sequence identical to that of the natural molecule. Conformation- dependent immunologic assays indicate that the recombinant protein is produced with the native conformation.