title: Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5.
abstract: BACKGROUND
  Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein.
  
  
  OBJECTIVE
  To clone and characterize timothy grass pollen allergen Phl p 4.
  
  
  METHODS
  Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies.
  
  
  RESULTS
  Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner.
  
  
  CONCLUSIONS
  Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.
authors.affiliation: Pharmacia Diagnostics AB, Uppsala, Sweden.
authors.forename: A Marknell
authors.initials: AM
authors.lastname: Dewitt
authors.forename: K
authors.initials: K
authors.lastname: Andersson
authors.forename: G
authors.initials: G
authors.lastname: Peltre
authors.forename: J
authors.initials: J
authors.lastname: Lidholm
mesh_terms: D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D052179:Antigens, Plant; D000077102:Apium; D001483:Base Sequence; D019149:Bioreactors; D003001:Cloning, Molecular; D004306:Dose-Response Relationship, Immunologic; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D007526:Isoelectric Point; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology*; D006109:Poaceae; D011058:Pollen; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D017385:Sequence Homology
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C477756:Api g 5 allergen, celery; C100980:Phl p 4 protein, Phleum pratense; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E