ABSTRACT
Recombinant production in bacteria of soluble and monomeric Phl p 1, a major allergen of Timothy grass pollen, has proved to be very problematic. In order to facilitate expression and purification of this allergen, a recombinant variant was designed with a single amino acid substitution. Several comparative analyses with natural counterparts using electrophoretic and HPLC separations, together with immunological assays, demonstrated high equivalence. This is the first description of an approach aiming at an improvement of a natural like recombinant allergen.
Subject(s)
Allergens/metabolism , Plant Proteins/metabolism , Allergens/chemistry , Allergens/genetics , Amino Acid Sequence , Chromatography, High Pressure Liquid , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Immunohistochemistry , Molecular Sequence Data , Molecular Weight , Peptide Mapping , Plant Proteins/chemistry , Plant Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
The recombinant major grass pollen allergen Phl p 6 has been expressed with a N-terminal 6 x His-tag sequence and subsequently purified using nickel-chelating Sepharose. After cleavage of the tag-sequence, a second pass over the affinity chromatography revealed that even untagged rPhl p 6 bound tightly. In order to determine if that property is typical for Phl p 6, the natural allergen was purified in the same way starting with a grass pollen extract. Indeed, nPhl p 6 could be highly enriched in one step using nickel-chelating Sepharose. In addition to this new powerful purification method, the results provide further information in that the recombinant and natural allergens share a lot of properties, since biochemical characteristics are reflected in the purification strategies. The preparations of natural and recombinant Phl p 6 were used for comparative electrophoretic, chromatographic and immunological analysis which demonstrated high similarity.
