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 pPICZ alpha a, 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.
Hiller
K M
KM
Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill 27514-7290, USA.
Lubahn
B C
BC
Klapper
D G
DG
D000485:Allergens / Q000235:genetics*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D017343:Genes, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics*
D000485:Allergens; C038640:Amb a VI protein, Ambrosia artemisiifolia; D052179:Antigens, Plant; D018076:DNA, Complementary; D010940:Plant Proteins