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Development of immunoaffinity chromatographic method for Ara h 2 isolation
- Source :
- Protein Expression and Purification. 131:85-90
- Publication Year :
- 2017
- Publisher :
- Elsevier BV, 2017.
-
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.
- Subjects :
- 0301 basic medicine
Arachis
High selectivity
01 natural sciences
Antibodies
Chromatography, Affinity
Sepharose
03 medical and health sciences
Affinity chromatography
Coupling efficiency
Glycoproteins
Chromatography
biology
Chemistry
010401 analytical chemistry
Extraction (chemistry)
food and beverages
Antigens, Plant
biochemical phenomena, metabolism, and nutrition
0104 chemical sciences
carbohydrates (lipids)
030104 developmental biology
Polyclonal antibodies
biology.protein
Target protein
2S Albumins, Plant
Biotechnology
Subjects
Details
- ISSN :
- 10465928
- Volume :
- 131
- Database :
- OpenAIRE
- Journal :
- Protein Expression and Purification
- Accession number :
- edsair.doi.dedup.....fa98a48e38536f5045886cdf1dfde202