Parvalbumin from carp, a major allergen,was purified to homogeneity using ion exchange chromatography and size exclusion chromatography (estimated purity \u3e 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
