EVALUATION OF THE ACTION OF PRECIPITATING AGENTS TO OBTAIN ALLERGENIC PROTEINS FROM CRUDE MITE EXTRACTS

Introduction: Allergen proteins found in dust mite extracts, such as Dermatophagoides farinae (DF), Dermatophagoides pteronyssinus (DP) and Tyrophagus putrescentiae (TP), are relevant for scientific studies in the allergy and immunotherapy fields. The precipitation/concentration of protein extracts may favor the aggregation of the allergens in homogenates. Method: This paper investigates the precipitation process by submitting crude mite extracts to compounds such as ammonium sulfate (NH4)2SO4, trichloroacetic acid (TCA) and acetone. Results: The best results were obtained by fractionation with (NH4)2SO4 at 80% (w/v) saturation (~0° C), observing the protein markings on the electrophoresis gel. Major allergens were identified by immunoblot at 25 kDa (cysteine protease) for Der f and Der p; and 25 kDa, 30 kDa (tropomyosin) and Try p 3, near 26 kDa. For this percentage the total protein contents were 12.83 mg mL-1 for Der f, 24.78 mg mL-1 for Der p and 27.35 mg mL-1 for Try. Conclusion: An advantage of precipitation with (NH4)2SO4 over precipitation with acetone was the possibility of gradually obtaining protein fractions, which does not happen when using the latter. The addition of 80% (v/v) acetone to the mite extracts favored total protein precipitation in the concentrations 16.42 mg mL-1, 28.47 mg mL-1 and 13.41 mg mL-1. The use of TCA in concentrations above 20% (w/v) forms peptides that are not retained in the gel under the established experimental conditions, and dilute solutions of this acid are more efficient.