title: Ani s 1, the major allergen of Anisakis simplex: purification by affinity chromatography and functional expression in Escherichia coli.
abstract: Third stage larvae of the nematode Anisakis simplex often infect marine fish and invertebrates. When the larvae are ingested orally via seafood, they can cause IgE-mediated allergic reactions as well as anisakiasis. Of the known A. simplex allergens, Ani s 1 (Kunitz/bovine pancreatic trypsin inhibitor family protein) has been demonstrated to be a major allergen, being expected to be a useful tool for diagnosis of A. simplex allergy. For a diagnostic purpose, sufficient amounts of either natural Ani s 1 (nAni s 1) or recombinant Ani s 1 (rAni s 1) with an IgE-binding capacity should be stably supplied whenever needed. In this study, therefore, we first developed a simple and rapid purification method for Ani s 1 that is based on affinity chromatography using anti-Ani s 1 antibodies as ligands. The method was shown to produce nAni s 1 with a higher yield than the previously reported methods. Then, an attempt was made to express rAni s 1 in Escherichia coli as a His-tagged protein. rAni s 1 obtained as an inclusion body was solubilized in a solvent containing denaturing and reducing reagents and purified by nickel-chelate chromatography. Refolding of rAni s 1 was accomplished by dialysis in the presence of arginine, followed by that in the absence of arginine. Fluorescence ELISA and inhibition ELISA data revealed that rAni s 1 is IgE reactive enough to be used as a diagnostic tool.
authors: Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo, Japan.
authors: Yukihiro
authors: Y
authors: Kobayashi
authors: Shoichiro
authors: S
authors: Ishizaki
authors: Yuji
authors: Y
authors: Nagashima
authors: Kazuo
authors: K
authors: Shiomi
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D017129:Anisakiasis / Q000276:immunology* / Q000469:parasitology; D017163:Anisakis / Q000276:immunology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D006967:Hypersensitivity / Q000175:diagnosis / Q000276:immunology; D007073:Immunoglobulin E / Q000097:blood; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000485:Allergens; D002135:Calcium-Binding Proteins; D015801:Helminth Proteins; D011994:Recombinant Proteins; C412511:troponin-like protein, Anisakis simplex; D007073:Immunoglobulin E