Cloning and Expression of Ragweed Allergen Amb a 6



We have cloned the protein coding region of an isoform of short ragweed allergen Amb a 6 (Ra6) and expressed the secreted product in Pichia pastoris at mg/l levels. 5′ RACE was performed using sequence obtained from a partial Amb a 6 clone. This yielded a product whose deduced protein sequence has a characteristic signal sequence motif at the N-terminus followed by sequence consistent with that previously published for highly purified Amb a 6 [Roebber et al. J Immunol 1983;131:706–11]. The region encoding the secreted product was amplified by PCR and cloned into pPICZαa, an expression vector for the yeast Pichia pastoris. Yeast transformed with this vector secrete a protein which migrates near Amb a 6 in SDS–PAGE. This secreted protein reacts with polyclonal anti-Amb a 6 antisera as well as an anti-Amb a 6 monoclonal antibody, and has the N-terminal sequence of Amb a 6. By time-of-flight mass spectrometry, recombinant Amb a 6 has a molecular weight of 9884 ± 0.2%. In addition to the deduced amino acid sequence of an Amb a 6 clone, the amino acid sequence of Amb a 6 protein is reported for comparison. The amino acid sequence was obtained by aligning overlapping tryptic and chymotryptic peptides from enzymatic digests of extensively reduced and alkylated Amb a 6. Sequences from at least three closely related Amb a 6 isoforms are present among these peptides. The amino acid sequence closely matches the deduced amino acid sequence of the Amb a 6 clone.