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Purification of Parvalbumin from Carp: A Protocol that Avoids Heat‐Treatment
Author(s) -
Koppelman Stef J.,
Romijn Roland A.,
De Jongh Harmen H.J.,
Nordlee Julie A.,
Piersma Sander,
Hessing Martin,
Taylor Steve L.
Publication year - 2010
Publication title -
journal of food science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.772
H-Index - 150
eISSN - 1750-3841
pISSN - 0022-1147
DOI - 10.1111/j.1750-3841.2010.01513.x
Subject(s) - parvalbumin , carp , chromatography , amino acid , chemistry , circular dichroism , biochemistry , biology , allergen , fish <actinopterygii> , allergy , immunology , genetics , fishery
Parvalbumin from carp, a major allergen, was purified to homogeneity using ion exchange chromatography and size exclusion chromatography (estimated purity >95% to 98% based on SDS‐PAGE and native PAGE) with a yield of 318 mg, and a number of basic biochemical characteristics were determined. The identity was confirmed by peptide‐mass fingerprinting, and IgE‐binding was demonstrated. The UV/Vis absorbance spectra were explained using the previously published amino acid sequences. Far UV‐CD spectroscopy was used to confirm the folding character of parvalbumin. We conclude that parvalbumin from carp can be purified on a comparatively large (hundreds of milligrams) scale using a purification protocol that does not include denaturing steps. The purified protein resembles biochemical characteristics as were earlier published for carp parvalbumin, that is, a molecular weight of approximately 12 kDa, amino acid sequence identity and a secondary structure containing α‐helices and β‐structures. The described method provides a yield sufficient to produce and characterize antibodies to construct immunochemical methods to detect parvalbumin in food, as well as for use as a standard calibrator for such assays. Practical Application: Parvalbumin is a major allergen from fish. Here, we have purified a comparatively large quantity from carp that can be used to develop antisera for use in an assay method to detect fish allergens.