Cloning, expression and characterization of the major latex allergen prohevein

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 Prohevei...

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Bibliographic Details
Published in:Clinical and experimental allergy 1998-11, Vol.28 (11), p.1418-1426
Main Authors: ROZYNEK, P, POSCH, A, BAUR, X
Format: Article
Language:English
Subjects:
Allergens - genetics
Allergens - immunology
Allergic diseases
Amino Acid Sequence
Antibody Specificity
Antigens, Plant
Antimicrobial Cationic Peptides
ATP-Binding Cassette Transporters
Base Sequence
Biological and medical sciences
Carrier Proteins - genetics
Cloning, Molecular
DNA, Complementary - chemistry
DNA, Complementary - genetics
Escherichia coli Proteins
Gene Expression
General aspects
Hev b 6.01
IgE
Immunoblotting
Immunoglobulin G - immunology
Immunopathology
Latex - immunology
Latex - metabolism
latex allergy
Lectins - immunology
Maltose-Binding Proteins
Medical sciences
Molecular Sequence Data
Monosaccharide Transport Proteins
Plant Lectins
Plant Proteins - genetics
Plant Proteins - immunology
prohevein
Protein Precursors - genetics
Protein Precursors - immunology
Recombinant Fusion Proteins - analysis
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - immunology
Sequence Analysis, DNA
Sequence Homology, Amino Acid
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Summary: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.
ISSN:0954-7894
1365-2222
DOI:10.1046/j.1365-2222.1998.00422.x