High-Level Expression in Mammalian Cells of Recombinant House Dust Mite Allergen ProDer p 1 with Optimized Codon Usage

Abstract

Background: The major house dust mite allergen Der p 1 is associated with allergic diseases such as asthma. Production of recombinant Der p 1 was previously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer p 1, a recombinant precursor form of Der p 1, in CHO cells. Methods: As optimization of the codon usage may allow successful overexpression of protein in mammalian cells, a synthetic gene encoding ProDer p 1 was designed on the basis of the codon usage frequently found in highly expressed human genes. Gene synthesis was accomplished from a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction. Results: COS cells transiently transfected with the synthetic ProDer p 1 gene produced up to 5–10 times as much ProDer p 1 compared with the expression level obtained after transfection with the authentic gene. To stably express the recombinant allergen, CHO-K1 cells were transfected with the ProDer p 1 synthetic gene, and one amplified recombinant clone produced up to 30 mg of recProDer p 1 per liter in the culture medium before purification. recProDer p 1 was secreted as an enzymatically inactive single-chain molecule presenting three glycosylated immunoreactive forms of 41, 38 and 36 kD. When examined with respect to direct binding, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivities. However, IgE inhibition and histamine release assays showed a much higher reactivity to natural Der p 1 compared to recProDer p 1. Conclusions: These data indicated that codon optimization represents an attractive strategy for high-level production of allergen in mammalian cells.

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

“…15 In these constructions, the potential N-glycosylation sites in the propeptide (N16p) and/or in the protease domain (N52) were eliminated by substitution of asparagine by glutamine (N16pQ, N52Q). Wild-type (WT) ProDer p 1 and the three mutants were successfully secreted by P. pastoris.…”

“…15 The sequences of the primers used in this study are detailed in Table 2. The amplified fragments were cloned into the pCR2.1 TOPO cloning vector (Invitrogen, Groeningen, The Netherlands), and the presence of both mutations of interest was verified by DNA sequencing.…”

“…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.…”

The House Dust Mite Allergen Der p 1, Unlike Der p 3, Stimulates the Expression of Interleukin-8 in Human Airway Epithelial Cells via a Proteinase-activated Receptor-2-independent Mechanism

“…Recombinant ProDer p 1 from CHO spent culture medium was purified to homogeneity as previously described (26). Before use, natural Der p 1 was activated for 15 min at room temperature in PBS, pH 7.3, containing 10 mM L-cysteine.…”

Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from <i>Dermatophagoides pteronyssinus</i>

“…As such, attempts to express the mature protein in E. coli led to poor yields and altered conformation of the molecule [14]. Furthermore, only proDer p 1 forms have been successfully expressed in various eukaryotic systems such as plants, insect and mammalian cells or the methanotrophic yeast Pichia pastoris [15,16,17,18,19,20], implying that the propeptide region is absolutely required for Der p 1 expression in eukaryotic systems. Such recombinant proforms of Der p 1 exhibit IgE reactivity and can be processed into active mature forms [18,21,22,23].…”