Art v 1 isoforms: Cloning, E.coli expression and immunological analysis

Abstract

Objective To examine the sequence polymorphism of Art v 1, the major allergen of mugwort pollen. Methods After screening of a mugwort pollen lambda ZAP II cDNA library with antibodies and DNA probes, forty cDNA clones coding for Art v 1 were isolated and sequenced. For the expression in competent E.coli strain BL 21 (+RIL), the cDNAs without the natural leader sequence were cloned into the expression plasmid pHis2. The immunoreactivity of purified Histagged recombinant isoforms was tested by immunoblots with a polyclonal rabbit serum raised against rArt v 1, a moAb anti-Art v 1 produced by genetic immunization and sera from 16 subjects allergic to mugwort pollen. Results The derived protein sequences of 7 different isoforms (corresponding to 25 different cDNAs) showed amino acid sequence identities ranging from 94% (6 amino acids exchanges) to 99% (a single amino acid exchange). Interestingly, all amino acid exchanges in the isoforms were restricted to the C-terminal hydroxyproline-rich domain, which carries the O-glycans of Art v 1. The mature isoforms were expressed in E.coli at a concentration of approximately 1 mg/L culture, depending on the isoform. All seven recombinant Art v 1 isoforms displayed similar IgG- and IgE-binding capacity. Conclusion Production of glycosylated recombinant isoforms in a plant-based expression system will help to clarify if the amino acid exchanges result in differences of the glycosylation pattern and antibody binding properties of Art v 1.