Maturation of Pichia pastoris‐derived recombinant pro‐Der p 1 induced by deglycosylation and by the natural cysteine protease Der p 1 from house dust mite

The mature cysteine protease from Dermatophgoides pteronyssinus, Der p 1, is a major house dust mite allergen. Its enzymatic activity has been shown to have pro‐inflammatory effects that could also negatively influence efficacy of allergen‐specific immunotherapy. The aim of this study was to express recombinant pro‐Der p 1 (rpro‐Der p 1) in the yeast Pichia pastoris and to study its maturation. Expression was achieved at a concentration ranging from 45 mg·L−1 (methanol‐induced expression) to 168 mg·L−1 (constitutive expression). No significant spontaneous maturation of the secreted proenzyme was observed. rpro‐Der p 1 with a sequence‐based molecular mass of 34 kDa was hyperglycosylated by the yeast, migrating at 50–60 kDa on SDS/PAGE. Compared with its natural counterpart (nDer p 1), the recombinant proenzyme demonstrated decreased IgE reactivity, resulting in a 30‐fold lower capacity to induce histamine release from human basophils. Decreased immunoreactivity was also shown by competitive RIA and sandwich ELISA with Der p 1‐specific antibody reagents. CD spectra of rpro‐Der p 1 and nDer p 1 revealed significant structural differences. Deglycosylation of rpro‐Der p 1 with endoglycosidase H resulted in a decrease in apparent molecular mass from 50 kDa to 34 kDa, but did not affect nDer p 1. On removal of N‐glycans from rpro‐Der p 1, which harbours two putative N‐glycosylation sites in both propeptide and mature sequence, the mature rDer p 1 appeared. This suggests that hyperglycosylation hampers spontaneous maturation. Maturation of the recombinant pro‐enzyme was also achieved by addition of the active natural cysteine protease, nDer p 1. In conclusion, high‐level expression of rpro‐Der p 1 in P. pastoris results in a stable hypoallergenic proenzyme with potential for use in allergen‐specific immunotherapy.