Purification of Parvalbumin from Carp: A Protocol That Avoids Heat Treatment

Authors

• Stef J. Koppelman, University of Nebraska-Lincoln and HAL Allergy, Leiden, NetherlandsFollow
• Roland A. Romijn, U-Protein Express, Utrecht, Netherlands
• Harmen H. J. de Jongh, Top Institute Food and Nutrition, Wageningen, NetherlandsFollow
• Julie A. Nordlee, University of Nebraska-LincolnFollow
• Sander Piersma, Vrije Universiteit Medical Center, Amsterdam, NetherlandsFollow
• Martin Hessing, U-Protein Express, Utrecht, Netherlands
• Steve L. Taylor, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

2010

Citation

Journal of Food Science (2010) 75(3): T49–T56

doi: 10.1111/j.1750-3841.2010.01513.x

Comments

Copyright © 2010, Institute of Food Technologists. Published by Wiley. Used by permission

Abstract

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.