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 binding of human IgE antibodies to Sol i 2 has been demonstrated to be conformation-dependent.


METHODS
A couple 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 III transfer vector (Invitrogen Corp., San Diego, Calif.), and the recombinant baculovirus was isolated by plaque purification. The recombinant baculovirus was grown in Sf9 and High-Five cells (Invitrogen Corp.) in serum-free media. The recombinant Sol i 2 was isolated and characterized.


RESULTS
Recombinant (r) Sol i 2 was produced in microgram/per milliliter amounts in Sf9 cells and at 30 micrograms/ml in High-Five cells. It was isolated by ultrafiltration and reverse-phase chromatography. The rSol i 2 demonstrated similar binding to natural-Sol i 2 in both a conformation-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 identical to that of the natural molecule. No significant increase in binding activity was found after treatment of rSol i 2 with protein disulfide isomerase. The binding of rSol i 2 to a conformation-dependent monoclonal antibody was lost by heating in sodium dodecylsulfate and reduction.


CONCLUSIONS
A recombinant Sol i 2 protein was produced at high yield in a baculovirus expression system by using serum-free medium with a sequence identical to that of the natural molecule. Conformation-dependent immunologic assays indicate that the recombinant protein is produced with the native conformation.
Schmidt
M
M
Department of Laboratory Medicine, Karolinska Hospital, Stockholm, Sweden.
McConnell
T J
TJ
Hoffman
D R
DR
D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D000862:Ant Venoms / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000937:Antigen-Antibody Reactions; D016367:Baculoviridae / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000276:immunology; D007535:Isomerases / Q000494:pharmacology; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification
D000485:Allergens; D000862:Ant Venoms; D011994:Recombinant Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases