Dataset Information

Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1.

ABSTRACT: Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.

SUBMITTER: Kaeswurm JAH

PROVIDER: S-EPMC7838873 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1.

Kaeswurm Julia A H JAH Nestl Bettina B Richter Sven M SM Emperle Max M Buchweitz Maria M

Methods and protocols 20201227 1

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent <i ...[more]

PMID: 33375510

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Dataset Information

Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1.

Publications

Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1.

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