Cloning, expression and characterization of the major latex allergen prohevein

Background About 70-80% of latex allergic health care workers are sensitize d 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 immun ochemical characterization of rHev b 6.01. Methods Prohevein was expressed in the periplasmatic space of Escherichia c oli as maltose binding protein (MBP) fusion protein and purified to homogen eity after factor Xa cleavage. The IgE binding capacity of both rHev b 6.01 and prohevein isolated from fresh Hevea latex was compared by immunoblotti ng 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 dedu ced amino acid sequence of both clones revealed two and three amino acid di fferences 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 u sing sera of affected subjects binding intensity to both proteins was compa rable 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 di agnostic 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.