Premium
Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2
Author(s) -
Suck Roland,
Petersen Arnd,
Weber Bernhard,
Becker WolfMeinhard,
Fiebig Helmut,
Cromwell Oliver
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305697
Subject(s) - recombinant dna , chromatography , allergen , polyacrylamide gel electrophoresis , chemistry , gel electrophoresis , capillary electrophoresis , high performance liquid chromatography , western blot , biology , microbiology and biotechnology , biochemistry , enzyme , immunology , allergy , gene
Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high‐resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N ‐terminal sequences and IgE‐binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.