title: Combination of zymography and immunodetection to analyze proteins in complex culture supernatants.
abstract: The physiological function of an allergen might be an important factor for the allergenicity. The major grass pollen allergen Phl p 1 shows sequence similarities to the consensus sequences of cysteine proteases. However, up to now, the proteolytic activity of Phl p 1 is controversial. The culture supernatant of Phl p 1-transfected clones from Pichia pastoris showed a proteolytic activity but this might be due to Phl p 1 or irrelevant yeast contaminants. To solve this question, we made use of the zymogram technique and improved it. Substrate as well as substrate concentration was changed from 1% casein to 0.25% skimmed milk powder. For staining, we used a colloidal Coomassie stain (RotiBlue) with a higher sensitivity and better practicability than the conventional Coomassie staining. The proteins in the zymogels and in the SDS-PAGE gels showed similar electrophoretic mobility. Furthermore, the zymogels could be blotted and immunostained. Thus, the molecular mass of the proteolytic bands could be determined and directly compared with immunoblotting results. To clearly assign the protease, we separated the culture supernatant of the Phl p 1-transfected P. pastoris clone by affinity chromatography with monoclonal antibody. Our studies demonstrate that the proteolytic activity did not belong to the recombinant allergen but to the yeast proteins. The enzyme was classified by zymogram inhibition tests as a strong serine protease.
authors: Research Center Borstel, Biochemical and Molecular Allergology, Borstel, Germany.
authors: Marco
authors: M
authors: Pöppelmann
authors: Wolf-Meinhard
authors: WM
authors: Becker
authors: Arnd
authors: A
authors: Petersen
mesh_terms: D000485:Allergens; D015153:Blotting, Western; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010940:Plant Proteins / Q000032:analysis*; D011506:Proteins / Q000032:analysis*
chemical_list: D000485:Allergens; D003470:Culture Media; D010940:Plant Proteins; D011506:Proteins; C090396:PHLPI protein, Phleum pratense