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

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 ∼1% of the total cellular protein in infected Sf9 insect cells. The protein was purified using Ni 2+ affinity column chromatography and the yield of purified protein was ∼10 mg/1g 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45340/1/11010_2004_Article_5141584.pd

Use of a Synthetic Peptide Epitope of Asp f 1, a Major Allergen or Antigen of Aspergillus fumigatus, for Improved Immunodiagnosis of Allergic Bronchopulmonary Aspergillosis

Allergic bronchopulmonary aspergillosis (ABPA) is an immunologically complex allergic disorder caused by the fungal pathogen Aspergillus fumigatus. Elevated levels of total immunoglobulin E (IgE), specific IgE, and IgG antibodies in sera are important immunodiagnostic criteria for ABPA. International reference standards or standardized immunodiagnostic assays are not available due to a lack of well-defined diagnostic antigens. The present study was carried out to identify and evaluate the immunodiagnostic relevance of synthetic epitopic peptides of Asp f 1, a major allergen, antigen, or cytotoxin of A. fumigatus. Five overlapping peptides were synthesized from the N terminus of Asp f 1, one of the potential immunodominant regions predicted by algorithmic programs. The 11-amino-acid synthetic peptide (P1) significantly inhibited both IgG binding (89.10% ± 4.45%) and IgE binding (77.32% ± 3.38%) of the standardized diagnostic antigen (SDA) (a well-defined pool of diagnostically relevant allergens and antigens of A. fumigatus). With a panel of sera of ABPA patients, allergic patients with skin test negativity to A. fumigatus, and healthy individuals, P1 showed a higher diagnostic efficiency than SDA (specific IgG, 100%; specific IgE, 98.3%). The diagnostic efficiency of P1 could be attributed to the presence of homologous epitopes in various immunodominant allergens or antigens of A. fumigatus. The ability of P1 to induce histamine release from sensitized mast cells and a Th2 type of cytokine profile in peripheral blood mononuclear cells of ABPA patients suggests its potential for use in intradermal testing. P1 could be further explored for development of a standardized, specific, and sensitive immunodiagnostic test for aspergillosis

Discovery of a Potent and Orally Efficacious TGR5 Receptor Agonist

TGR5 is a G protein-coupled receptor (GPCR), activation of which promotes secretion of glucagon-like peptide-1 (GLP-1) and modulates insulin secretion. The 2-thio-imidazole derivative <b>6g</b> was identified as a novel, potent, and selective TGR5 agonist (hTGR5 EC₅₀ = 57 pM, mTGR5 = 62 pM) with a favorable pharmacokinetic profile. The compound <b>6g</b> was found to have potent glucose lowering effects <i>in vivo</i> during an oral glucose tolerance test in DIO C57 mice with ED₅₀ of 7.9 mg/kg and ED₉₀ of 29.2 mg/kg

Discovery of a Potent and Orally Efficacious TGR5 Receptor Agonist

TGR5 is a G protein-coupled receptor (GPCR), activation of which promotes secretion of glucagon-like peptide-1 (GLP-1) and modulates insulin secretion. The 2-thio-imidazole derivative <b>6g</b> was identified as a novel, potent, and selective TGR5 agonist (hTGR5 EC₅₀ = 57 pM, mTGR5 = 62 pM) with a favorable pharmacokinetic profile. The compound <b>6g</b> was found to have potent glucose lowering effects <i>in vivo</i> during an oral glucose tolerance test in DIO C57 mice with ED₅₀ of 7.9 mg/kg and ED₉₀ of 29.2 mg/kg