title: Biochemical and immunological characterization of a recombinant precursor form of the house dust mite allergen Der p 1 produced by Drosophila cells.
abstract: BACKGROUND
  The major house dust mite allergen Der p 1 elicits strong IgE antibody responses in patients suffering from mite allergy.
  
  
  OBJECTIVE
  This study reports the expression and characterization of a recombinant precursor form of Der p 1 secreted as ProDer p 1 from insect cells.
  
  
  METHODS
  The cDNA coding for ProDer p 1 was cloned downstream to the gp67 signal peptide, starting from commercial cDNA encoding Der p 1 and PCR-amplified ProDer p 1 genomic fragment. ProDer p 1, expressed in Drosophila cells and purified from culture medium, was compared to Der p 1 isolated from mite culture, in terms of glycosylation, enzymatic activity as well as IgG- and IgE-binding capacity.
  
  
  RESULTS
  Sequence analysis of the genomic clone of ProDer p 1 revealed that, besides two introns in the mature Der p 1 coding sequence, two introns were also present in the propeptide coding sequence. ProDer p 1 was purifed to homogeneity by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. The precursor form of Der p 1 could be processed in vitro into mature Der p 1 under acidic and reducing conditions. Carbohydrate analysis clearly indicated that ProDer p 1 expressed from insect cells was glycosylated and that glycan structures were located only in the prosequence. ProDer p 1 displayed a similar immunoreactivity towards IgE, monoclonal and polyclonal IgG antibodies compared to natural Der p 1. Specific activity measurements using synthetic substrates clearly indicated that, contrary to natural Der p 1, ProDer p 1 was totally enzymatically inactive.
  
  
  CONCLUSIONS
  The expression of an enzymatically inactive and highly antigenic ProDer p 1 zymogen molecule could be a suitable strategy for the development of in vitro diagnosis test as well as for specific immunotherapy.
authors.affiliation: Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Jacquet
authors.forename: M
authors.initials: M
authors.lastname: Haumont
authors.forename: M
authors.initials: M
authors.lastname: Massaer
authors.forename: V
authors.initials: V
authors.lastname: Daminet
authors.forename: L
authors.initials: L
authors.lastname: Garcia
authors.forename: P
authors.initials: P
authors.lastname: Mazzu
authors.forename: P
authors.initials: P
authors.lastname: Jacobs
authors.forename: A
authors.initials: A
authors.lastname: Bollen
mesh_terms: D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides; D001483:Base Sequence; D003001:Cloning, Molecular; D004330:Drosophila / Q000235:genetics*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D008925:Mites / Q000737:chemistry* / Q000201:enzymology / Q000276:immunology*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D011499:Protein Processing, Post-Translational / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology*; D017422:Sequence Analysis, DNA
chemical_list: D000485:Allergens; D039741:Antigens, Dermatophagoides; D004792:Enzyme Precursors; D006023:Glycoproteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D007073:Immunoglobulin E