Recombinant Alternaria alternata ENO was expressed in E. coli.
Alt a 2 (Alt a 6 according to the effective allergen nomenclature) is a lyophilized, recombinant protein with IgE-binding capacity. It was produced by heterologous expression in E. coli, purified by conventional biochemical methods.
Enolase activity can be restored by dissolving the lyophilized protein to a concentration of 1 mg/ml in 4.5 M urea and dialyzing it over night against 10mM NaxHxPO4 pH 7.5, containing 2mM -Mercaptoethanole, and 2mM Mg2+.No enzymatic activity was observed after reconstitution in water/ -Mercaptoethanole.
When stored at -20 centigrade the quality of the material will be maintained for several years. However, for short periods (max. 3 weeks) the lyophilized product may be kept at room temperature. After reconstitution store at -20 centigrade. Avoid repeated freezing/thawing.
The material can be reconstituted with distilled water (or equivalent) or dilute buffers. Addition of 1-2 mM -Mercaptoethanol is recommended. If reconstituted to a concentration of 1 mg/ml, the phosphate buffer concentration will be 2 mM (pH 7,4). The product is soluble to more than 95% at a concentration of 2 mg/ml.