Purification of Parvalbumin from Carp: A Protocol that Avoids Heat-Treatment

Stef J. Koppelman, Roland A. Romijn, Harmen H.J. de Jongh, Julie A. Nordlee, Sander Piersma, Martin Hessing, Steve L. Taylor

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)T49-T56
JournalJournal of food science
Volume75
Issue number3
DOIs
StatePublished - Apr 2010

Keywords

  • Allergen
  • Carp
  • Fish
  • Parvalbumin

ASJC Scopus subject areas

  • Food Science

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