High‐level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris

Abstract

This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.

Relationship between Propeptide pH Unfolding and Inhibitory Ability during ProDer p 1 Activation Mechanism

“…This phenomenon was also reported for other cysteine proteases and has been observed for ProDer p 1 maturation. [12][13][14] These two cleavages occurred at sequences Asp76p-Leu77p-Asn78p and Asn78p-Ala79p-Glu80p and were in agreement with the Der p 1 substrate specificity. In other papain-like protease precursors, additional cleavages occurred in a region located just after the second β-strand.…”

“…However, surprisingly, for the N16pQ and N16pQ/N52Q mutants, partial activation occurred during the production, leading to the appearance of different forms. The N-terminal sequencing of the purified mutants revealed the presence of at least three forms, previously described by Takai et al and Jacquet et al 13,14 The first one displayed the ATFE sequence, which corresponds to the cleavage of the peptide bond between Tyr19p and Ala20p. This site is only three residues away from the propeptide Nglycosylation site and gives rise to the loss of the first α-helix of the propeptide (Fig.…”

“…The maturation was performed as previously described, 14 with the following modifications: the purified ProDer p 1 was dialyzed against 100 mM acetate buffer at pH 4 during 72 h at 4°C. 13,14 For the N52Q proform, maturation was performed at 37°C in polybuffer (mix of 50 mM Tris, phosphate, citrate, acetate and KCl, adjusted to chosen pH with HCl, pH 4), with or without the addition of 1 mM DTT and 1 mM EDTA.…”

“…[12][13][14] Despite several attempts, in vitro demonstration of the activation of ProDer p 1 remains partly unsuccessful. For example, purified recombinant proforms of Der p 1 expressed in mammalian CHO and insect Drosophila cells could be converted into mature forms by incubation at pH 4 but do not exhibit enzymatic activity.…”

“…15,16 More recently, however, several groups have succeeded in converting ProDer p 1 expressed in Pichia pastoris into mature enzymatically active forms with IgE reactivity. 13,14 Finally, several experiments performed with ProDer p 1 mutants lacking the N-glycosylation sites or with endoglycosylase H-pretreated ProDer p 1 indicated that the glycosylation of recombinant zymogen expressed in P. pastoris can impair the maturation of the allergen. 17,18 Analysis of the X-ray structure of the zymogen form of the allergen reveals that the propeptide of Der p 1 adopts a new fold within the CA1 protease family 19 ( Fig.…”