Molecular and immunological characterization of subtilisin like serine protease, a major allergen of Curvularia lunata.
Serine protease from numerous sources have been identified and characterized as major allergens. The present study aimed to clone, express and characterize a serine protease from Curvularia lunata. cDNA library screening identified partial protease clones. A clone showed significant homology to subtilisin like serine proteases from Aspergillus and Penicillium species. Full length sequence was generated by RACE PCR, subcloned in pET vector, protein expressed in Escherichia coli and purified from inclusion bodies yielding 0.5 mg/L of culture. Bioinformatic analysis identified serine protease motifs of subtilase family, catalytic triad and N-glycosylation sites on the primary sequence. The protein resolved at 54-kDa on SDS-PAGE and was recognized as a major allergen on immunoblot with 13/16 C. lunata sensitive patients' sera in ELISA and immunoblot. Recombinant protein reacted with rabbit polyclonal antibodies against alkaline serine proteases from C. lunata. Recombinant protein required 50-56 ng of same protein for 50% inhibition of IgE binding in competitive ELISA. In addition, 13 of 16 patients' samples showed significant basophil histamine release upon stimulation with purified recombinant protein. In conclusion, a 54 kDa major allergen of C. lunata was cloned, expressed, characterized and showed biological activity. It has potential to be used in molecule based approach for allergy diagnosis and therapy.
Tripathi
Prabhanshu
P
Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India.
Nair
Smitha
S
Singh
B P
BP
Arora
Naveen
N
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006636:Histamine Release; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020860:Subtilisin / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000485:Allergens; D007073:Immunoglobulin E; D057057:Serine Proteases; D020860:Subtilisin