RESUMEN
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.
Asunto(s)
Alérgenos/metabolismo , Proteínas de Plantas/metabolismo , Alérgenos/química , Alérgenos/genética , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
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.