title: Development of immunoaffinity chromatographic method for Ara h 2 isolation.
abstract: Ara h 2 is considered a major allergen in peanut. Due to the difficulty of separation, Ara h 2 had not been fully studied. Immunoaffinity chromatography (IAC) column can separate target protein with high selectivity, which made it possible to purify Ara h 2 from different samples. In this study, IAC method was developed to purify Ara h 2 and its effect was evaluated. By coupling polyclonal antibody (pAb) on CNBr-activated Sepharose 4B, the column for specific extraction was constructed. The coupling efficiency of the IAC column was higher than 90%, which made the capacity of column reached 0.56 mg per 0.15 g medium (dry weight). The recovery of Ara h 2 ranged from 93% to 100% for different concentrations of pure Ara h 2 solutions in 15 min. After using a column 10 times, about 88% of the column capacity remained. When applied to extract Ara h 2 from raw peanut protein extract and boiled peanut protein extract, the IAC column could recovery 94% and 88% target protein from the mixture. SDS-PAGE and Western blotting analysis confirmed the purified protein was Ara h 2, its purity reached about 90%. Significantly, the IAC column could capture dimer of Ara h 2, which made it feasible to prepared derivative of protein after processing.
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China. Electronic address: wuzhihua@ncu.edu.cn.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; School of Food Science & Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; School of Food Science & Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Shaode
authors.initials: S
authors.lastname: Zhan
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; School of Food Science & Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Lian
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; School of Food Science & Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Ruifang
authors.initials: R
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; School of Resources Environment & Chemical Engineering, Nanchang University, Nanchang 330047, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; School of Food Science & Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Tong
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; School of Food Science & Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China.
authors.forename: Anshu
authors.initials: A
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China. Electronic address: chbgjy@hotmail.com.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Chen
mesh_terms: D055516:2S Albumins, Plant / Q000737:chemistry* / Q000302:isolation & purification*; D000906:Antibodies / Q000737:chemistry*; D052179:Antigens, Plant / Q000737:chemistry* / Q000302:isolation & purification*; D010367:Arachis / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D055516:2S Albumins, Plant; D000906:Antibodies; D052179:Antigens, Plant; C500890:Ara h 2 allergen, Arachis hypogaea; D006023:Glycoproteins
keywords: Ara h 2; Immunoaffinity chromatography; Peanut; Polyclonal antibody