Supplementary Material for: A Recombinant Sal k 1 Isoform as an Alternative to the Polymorphic Allergen from <b><i>Salsola kali</i></b> Pollen for Allergy Diagnosis

<b><i>Background:</i></b> The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, <i>Salsola kali </i>is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of <i>S. kali </i>pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. <b><i>Methods:</i></b> Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) <i>Escherichia coli </i>cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from <i>S. kali </i>pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. <b><i>Results:</i></b> rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as <i>Platanus acerifolia</i> and Oleaceae members. <b><i>Conclusions:</i></b> rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.