High-level expression in mammalian cells of recombinant house dust mite allergen ProDer p 1 with optimized codon usage

Background: The major house dust mite allergen Der p 1 is associated with a llergic diseases such as asthma. Production of recombinant Der p 1 was prev iously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer pi, a recombinant precursor form of Der p 1, in CHO cells. Methods: As optimization of the codon usage may all ow successful overexpression of protein in mammalian cells, a synthetic gen e encoding ProDer p 1 was designed on the basis of the codon usage frequent ly found in highly expressed human genes. Gene synthesis was accomplished f rom a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction. Results: COS cells transiently transfec ted with the synthetic ProDer p 1 gene produced up to 5-10 times as much Pr oDer p 1 compared with the expression level obtained after transfection wit h the authentic gene. To stably express the recombinant allergen, CHO-K1 ce lls 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 cu lture medium before purification. recProDer p 1 was secreted as an enzymati cally inactive single-chain molecule presenting three;glycosylated immunore active forms of 41, 38 and 36 kD, When examined with respect to direct bind ing, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivit ies. However, IgE inhibition and histamine release assays showed a much hig her reactivity to natural Der pi compared to recProDer p 1. Conclusions: Th ese data indicated that codon optimization represents an attractive strateg y for high-level production of allergen in mammalian cells. Copyright (C) 2 001 S.Karger AG, Basel