HIGH-LEVEL EXPRESSION OF COCKROACH ALLERGEN, BLA-G-4, IN PICHIA-PASTORIS

Exposure to cockroach allergens is a risk factor for allergic disease and has been linked to an increase in asthma morbidity among cockroach -sensitive inner-city children. Bla g 4 is a ligand-binding protein (o r calycin) that causes IgE antibody responses in 40% to 60% of patient s allergic to cockroaches. Recombinant Bla g 4 was expressed in Escher ichia coli as an 18 kd protein but provided poor yields (only 0.25 mg/ L culture). To improve yields, Bla g 4 was expressed in the Pichia pas toris yeast system as a 23 kd secreted protein at concentrations of 50 mg allergen/L. By cross-inhibition radioimmunoassay, Bla g 4 expresse d in E. coli or P. pastoris. provided overlapping inhibition curves. B oth allergen preparations bound comparable Levels of serum IgE antibod y and showed similar skin test reactivity in individuals allergic to c ockroaches (10(-1) to 10(-3) mu g/ml). Deglycosylation of Pichia-expre ssed Bla g 4 with endoglycosidase F resulted in an 18 to 20 kd doublet , and liquid chromatography-mass spectrometry results suggested that t he 20 kd band contained residual sugar residues. Both glycosylated and deglycosylated Pichia Bla g 4 showed comparable inhibition of IgE ant ibody binding in radioimmunoassay. Pichia-produced Bla g 4 had the sam e antigenic reactivity as that produced in E. coli, and glycosylation had no effect on IgE antibody binding. The high yield of Bla g 4 obtai ned in the Pichia system will facilitate studies on the structure and function of calycin allergens and on the immune response of asthma pat ients to cockroach allergens.