TAILIEUCHUNG - Báo cáo khoa học: 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-in¯am-matory e ects that could alsonegatively in¯uence e cacy of allergen-speci®c immunotherapy. The aimof this study was to express recombinant pro-Der p 1 (rpro-Der p 1) in the yeastPichia pastorisand to study itsmaturation. | Eur. J. Biochem. 269 671-679 2002 FEBS 2002 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 Erica van Oort Pleuni G. de Heer W. Astrid van Leeuwen Ninotska I. L. Derksen Marcel Miiller Stephan Huveneers Rob C. Aalberse and Ronald van Ree CLB Department of Immunopathology and Laboratory for Experimental and Clinical Immunology Academic Medical Center University of Amsterdam the Netherlands 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 .

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