CLONING AND EXPRESSION OF RAGWEED ALLERGEN AMB A-6

We have cloned the protein coding region of an isoform of short ragwee d allergen Amba6 (Ra6) and expressed the secreted product in Pichia pa storis at mg/l levels. 5' RACE was performed using sequence obtained f rom a partial Amba6 clone. This yielded a product whose deduced protei n sequence has a characteristic signal sequence motif at the N-terminu s followed by sequence consistent with that previously published for h ighly purified Amb a6 [Roebber et al. J Immunol 1983,131:706-11]. The region encoding the secreted product was amplified by PCR and cloned i nto pPICZ alpha a, an expression vector for the yeast Pichia pastoris. Yeast transformed with this vector secrete a protein which migrates n ear Amb a 6 in SDS-PAGE. This secreted protein reacts with polyclonal anti-Amb a6 antisera as well as an anti-Amb a6 monoclonal antibody, an d has the N-terminal sequence of Amb a6. By time-of-flight mass spectr ometry, recombinant Amb a6 has a molecular weight of 9884 +/- 0.2%. In addition to the deduced amino acid sequence of an Amba6 clone, the am ino acid sequence of Amba6 protein is reported for comparison. The ami no acid sequence was obtained by aligning overlapping tryptic and chym otryptic peptides from enzymatic digests of extensively reduced and al kylated Amb a 6. Sequences from at least three closely related Amb a 6 isoforms are present among these peptides. The amino acid sequence cl osely matches the deduced amino acid sequence of the Amb a 6 clone.