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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Publications

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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Project description:BACKGROUND:The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic β-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation. RESULTS:rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. CONCLUSION:Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.

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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