A deglycosylated recombinant Der p 1 with improved mAb and IgE antibody binding activity

RATIONALE: Recombinant Derp1 expressed in Pichia pastoris has lower antibody binding activity than natural Derp1. Our goal was to investigate whether deglycosylation of Pichia expressed rDerp1 would produce an allergen that more closely resembles the activity of natural Derp1.METHODS: N-glycosylation sites in rDerp1 cDNA were removed by site-directed mutagenesis. Deglycosylated rDerp1was purified by affinity chromatography and compared to both glycosylated rDerp1 and nDerp1 by mAb ELISA, IgE Ab ELISA, and SDS-PAGE, before and after removal of the pro region by dialysis against 0.1M sodium acetate.RESULTS: mAb ELISA showed a 2.5 fold increase in reactivity with deglycosylated rDerp1 compared to glycosylated rDerp1 after removal of pro regions. SDS PAGE showed deglycosylated rDerp1 bands at 32kd and 25kd before and after pro region removal. Glycosylated rDerp1 bands appeared at 48kd and 37kd respectively.IgE Ab ELISA showed an 89% increase in reactivity of deglycosylated rDerp1 compared to glycosylated rDerp1. In addition, a strong correlation was found between IgE antibody binding to deglycosylated rDerp1 and nDerp1 (n = 72, r2 =0.90, p<0.001).CONCLUSIONS: Deglycosylation of rDerp1 expressed in Pichia, coupled with removal of the pro region, improved the immunoreactivity of rDerp1. This form of rDerp1 will be useful for allergen standardization and the development of improved allergy diagnostics and therapeutics. RATIONALE: Recombinant Derp1 expressed in Pichia pastoris has lower antibody binding activity than natural Derp1. Our goal was to investigate whether deglycosylation of Pichia expressed rDerp1 would produce an allergen that more closely resembles the activity of natural Derp1. METHODS: N-glycosylation sites in rDerp1 cDNA were removed by site-directed mutagenesis. Deglycosylated rDerp1was purified by affinity chromatography and compared to both glycosylated rDerp1 and nDerp1 by mAb ELISA, IgE Ab ELISA, and SDS-PAGE, before and after removal of the pro region by dialysis against 0.1M sodium acetate. RESULTS: mAb ELISA showed a 2.5 fold increase in reactivity with deglycosylated rDerp1 compared to glycosylated rDerp1 after removal of pro regions. SDS PAGE showed deglycosylated rDerp1 bands at 32kd and 25kd before and after pro region removal. Glycosylated rDerp1 bands appeared at 48kd and 37kd respectively. IgE Ab ELISA showed an 89% increase in reactivity of deglycosylated rDerp1 compared to glycosylated rDerp1. In addition, a strong correlation was found between IgE antibody binding to deglycosylated rDerp1 and nDerp1 (n = 72, r2 =0.90, p<0.001). CONCLUSIONS: Deglycosylation of rDerp1 expressed in Pichia, coupled with removal of the pro region, improved the immunoreactivity of rDerp1. This form of rDerp1 will be useful for allergen standardization and the development of improved allergy diagnostics and therapeutics.