Purification of parvalbumin from carp: a protocol that avoids heat-treatment.
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 alpha-helices and beta-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.


CONCLUSIONS
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.
Koppelman
Stef J
SJ
Food Allergy Research and Resource Program, Univ. of Nebraska, Lincoln, NE, USA. stefkoppelman@zonnet.nl
Romijn
Roland A
RA
de Jongh
Harmen H J
HH
Nordlee
Julie A
JA
Piersma
Sander
S
Hessing
Martin
M
Taylor
Steve L
SL
D000485:Allergens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D002347:Carps / Q000378:metabolism*; D002942:Circular Dichroism; D005374:Filtration / Q000379:methods; D029941:Fish Proteins / Q000302:isolation & purification*; D005512:Food Hypersensitivity / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010320:Parvalbumins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017747:Seafood; D016415:Sequence Alignment; D013053:Spectrophotometry; D013696:Temperature
D000485:Allergens; D029941:Fish Proteins; D010320:Parvalbumins; D007073:Immunoglobulin E