Expression and characterization of Asp fI, an immunodominant allergen/antigen of A. fumigatus in insect cell

• Published in: Molecular and cellular biochemistry 2003-10, Vol.252 (1-2), p.157-163
• Main Authors: Priyadarsiny, Priyanka, Swain, Prabodha K, Sarma, P Usha
• Format: Article
• Language: English
• Subjects: Allergens; Allergens - genetics; Allergens - immunology; Animals; Antigens; Antigens, Plant; Aspergillosis; Aspergillus fumigatus; Aspergillus fumigatus - immunology; Baculovirus; Base Sequence; Blotting, Western; Cloning, Molecular; Column chromatography; Cytotoxicity; Cytotoxins; DNA Primers; Electrophoresis, Polyacrylamide Gel; Expression vectors; Fungal Proteins - genetics; Fungal Proteins - immunology; Glycoproteins; Immunoglobulin E; Immunoglobulin G; Immunoreactivity; Insect cells; Insects; Monoclonal antibodies; Polymerase Chain Reaction; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Ribonuclease; Spodoptera - genetics; Translation
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1023/A:1025594620152

Asp fI is a major allergen/antigen/cytotoxin of Aspergillus fumigatus and exhibits ribonuclease activity. This allergen plays a role in allergic and invasive Aspergillosis and reported as a major cytotoxin with ribonuclease activity. To express the protein in large quantity and to characterize the multifunctional nature of Asp fI, we have generated recombinant baculovirus by introducing the gene in pFastBac HTa expression vector and expressed in insect cell. The baculovirus expression vector system has been used as a versatile system for the efficient expression of proteins with most eukaryotic posttranslational modification. Recombinant Asp fI was expressed as approximately 1% of the total cellular protein in infected Sf9 insect cells. The protein was purified using Ni2+ affinity column chromatography and the yield of purified protein was approximately 10 mg/l g of total cellular protein. Immunoreactivity of the protein was determined by immunoblot analysis using both poly His monoclonal antibody, IgG and IgE antibodies present in the sera of ABPA patients. The protein was glycosylated as revealed by the glycoprotein staining and was observed to retain both ribonuclease and cytotoxic activities. These results suggest that Asp fI expressed in insect cell was post translationally modified and biologically active that can be used as a diagnostic marker for biochemical studies.