tailieunhanh - Báo cáo khoa học: Cloning, expression and characterization of two new IgE-binding proteins from the yeast Malassezia sympodialis with sequence similarities to heat shock proteins and manganese superoxide dismutase

Malassezia sympodialisis an opportunistic yeast that col-onizes human skin and may induce IgE and T cell reac-tivity in patients with atopic eczema/dermatitis syndrome (AEDS). Previously, we have cloned and expressed six recombinant allergens (rMala s 1 and rMala s 5 to rMal-a s 9) from this yeast. By combining high throughput screening and phage surface display techniques, 27 com-plete and partial IgE-binding clones of M. sympodialis have been identified. Here we enlarged the panel of recombinant M. sympodialisallergens by RACE–PCR, cloning and nucleotide sequencing to obtain the coding sequences of two new IgE-binding clones. . | Eur. J. Biochem. 271 1885-1894 2004 FEBS 2004 doi Cloning expression and characterization of two new IgE-binding proteins from the yeast Malassezia sympodialis with sequence similarities to heat shock proteins and manganese superoxide dismutase Anna Andersson1 Omid Rasool1 Marait Schmidt2 Rimantas Kodzius3 Sabine Fliickiaer3 Arezou Zaraari1 Reto Crameri3 and Annika Scheynius1 1 Department of Medicine Clinical Allergy Research Unit Karolinska Institutet and Hospital Stockholm Sweden department of Biology and Department of Pathology and Laboratory Medicine East Carolina University Greenville NC USA 3 Swiss Institute of Allergy and Asthma Research SIAF Davos Switzerland Malassezia sympodialis is an opportunistic yeast that colonizes human skin and may induce IgE and T cell reactivity in patients with atopic eczema dermatitis syndrome AEDS . Previously we have cloned and expressed six recombinant allergens rMala s 1 and rMala s 5 to rMala s 9 from this yeast. By combining high throughput screening and phage surface display techniques 27 complete and partial IgE-binding clones of M. sympodialis have been identified. Here we enlarged the panel of recombinant M. sympodialis allergens by RACE-PCR cloning and nucleotide sequencing to obtain the coding sequences of two new IgE-binding clones. The coding sequences of one of the clones showed similarity to the heat shock protein HSP family and the other to manganese superoxide dismutase MnSOD and both had a high degree of homology to human counterparts. The coding sequences were expressed in Escherichia coli as six-histidine tagged recombinant proteins and generated products with molecular masses of kDa for HSP and kDa for MnSOD. Their IgE-binding frequencies were shown to be 69 and 75 respectively to 28 sera from AEDS patients with serum IgE to M. sympodialis extract indicating that HSP and MnSOD are major M. sympodialis allergens. In inhibition immunoblotting M. sympodialis extract

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