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Cloning and Expression of Ragweed Allergen Amb a 6
Author(s) -
Hiller Km,
BC Lubahn,
Klapper Dg
Publication year - 1998
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1046/j.1365-3083.1998.00355.x
Subject(s) - pichia pastoris , peptide sequence , biology , microbiology and biotechnology , polyclonal antibodies , signal peptide , pichia , amino acid , recombinant dna , antiserum , biochemistry , antibody , gene , genetics
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.