High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2.
Allergic reactions to peanuts are a serious health problem because of their high prevalence, associated with potential severity, and chronicity. One of the three major allergens in peanut, Ara h 2, is a member of the conglutin family of seed storage proteins. Ara h 2 shows high sequence homology to proteins of the 2S albumin family. Presently, only very few structural data from allergenic proteins of this family exist. For a detailed understanding of the molecular mechanisms of food-induced allergies and for the development of therapeutic strategies knowledge of the high-resolution three-dimensional structure of allergenic proteins is essential. We report a method for the efficient large-scale preparation of properly folded Ara h 2 for structural studies and report CD-spectroscopic data. In contrast to other allergenic 2S albumins, Ara h 2 exists as a single continuous polypeptide chain in peanut seeds, and thus heterologous expression in Escherichia coli was possible. Ara h 2 was expressed as Trx-His-tag fusion protein in E. coli Origami (DE3), a modified E. coli strain with oxidizing cytoplasm which allows the formation of disulfide bridges. It could be shown that recombinant Ara h 2, thus overexpressed and purified, and the allergen isolated from peanuts are identical as judged from immunoblotting, analytical HPLC, and circular dichroism spectra.
Lehmann
Katrin
K
Lehrstuhl Biopolymere, Universität Bayreuth 30, Universitaetsstrasse 30, 95440, Bayreuth, Germany
Hoffmann
Silke
S
Neudecker
Philipp
P
Suhr
Martin
M
Becker
Wolf-Meinhard
WM
Rösch
Paul
P
D055516:2S Albumins, Plant; D000818:Animals; D052179:Antigens, Plant; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000378:metabolism; D010940:Plant Proteins; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification
D055516:2S Albumins, Plant; D052179:Antigens, Plant; C078642:Ara h II protein, Arachis hypogaea; D006023:Glycoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E