title: Cloning, expression and characterization of the major latex allergen prohevein.
abstract: BACKGROUND
  About 70-80% of latex allergic health care workers are sensitized to prohevein (Hev b 6.01), a 20 kDa cysteine-rich chitin-binding protein of Hevea latex.
  
  
  OBJECTIVE
  This study reports on the bacterial cloning, expression and immunochemical characterization of rHev b 6.01.
  
  
  METHODS
  Prohevein was expressed in the periplasmatic space of Escherichia coli as maltose binding protein (MBP) fusion protein and purified to homogeneity after factor Xa cleavage. The IgE binding capacity of both rHev b 6.01 and prohevein isolated from fresh Hevea latex was compared by immunoblotting experiments using sera of latex-allergic patients. The diagnostic value of rHev b 6.01 was analysed by enzyme allergosorbent test (EAST).
  
  
  RESULTS
  Two different cDNA clones of rHev b 6.01 were established. The deduced amino acid sequence of both clones revealed two and three amino acid differences in the C-terminal domain of prohevein compared with the original database entry. Purified rHev b 6.01 bound with high affinity to chitin as its natural counterpart isolated from natural latex. In IgE-immunblotting using sera of affected subjects binding intensity to both proteins was comparable indicating a very high antigenic similarity. The diagnostic value of MBP-prohevein was tested in EAST using sera of 33 latex-allergic subjects. The in vitro test showed high sensitivity and specificity and proved the diagnostic value of uncleaved MBP-prohevein.
  
  
  CONCLUSIONS
  The production of recombinant latex key allergens with defined quality like prohevein is a straightforward strategy for the development of standardized in vitro test systems.
authors.affiliation: Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin, Institut an der Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Rozynek
authors.forename: A
authors.initials: A
authors.lastname: Posch
authors.forename: X
authors.initials: X
authors.lastname: Baur
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000485:Allergens / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D052179:Antigens, Plant; D023181:Antimicrobial Cationic Peptides; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D007840:Latex / Q000276:immunology / Q000378:metabolism*; D037102:Lectins / Q000276:immunology; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D037121:Plant Lectins; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology; D011498:Protein Precursors / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D018528:ATP-Binding Cassette Transporters; D000485:Allergens; D052179:Antigens, Plant; D023181:Antimicrobial Cationic Peptides; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; C097982:Hev b 6.01 protein, Hevea brasiliensis; D007074:Immunoglobulin G; D007840:Latex; D037102:Lectins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D037121:Plant Lectins; D010940:Plant Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C045875:hevein