Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2.
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.
Suck
Roland
R
Allergopharma Joachim Ganzer KG, Reinbek, Germany. roland.suck@allergopharma.de
Petersen
Arnd
A
Weber
Bernhard
B
Becker
Wolf-Meinhard
WM
Fiebig
Helmut
H
Cromwell
Oliver
O
D000485:Allergens / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000915:Antibody Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D007073:Immunoglobulin E; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D011058:Pollen / Q000276:immunology*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins; D016415:Sequence Alignment
D000485:Allergens; C084984:PHLPII protein, Phleum pratense; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D007073:Immunoglobulin E