Cloning and Expression of Ragweed Allergen Amb a 6

Abstract

Hiller KM, Lubahn BC, Klapper DG. Cloning and Expression of Ragweed Allergen Amb a 6. Scand J Immunol 1998;48:26-36 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 0 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 pPICZaa, 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 Ϯ 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.

The Spectrum of Allergens in Ragweed and Mugwort Pollen

“…Amb a 6 (formerly Ra6) is a basic protein with a molecular weight of 9.9 kDa [32]. It belongs to the ubiquitous group of plant nonspecific lipid transfer proteins (nsLTP) described as potent allergens in various fruits and as the major allergen of Parietaria pollen.…”

“…Nevertheless, they seem to have similar antigenic and allergenic properties when tested with hyperimmune animal antisera or IgE and IgG from Amb a 6-sensitized patients. One of the Amb a 6 isoforms was expressed as a secreted protein in Pichia pastoris and shown to react with polyclonal and monoclonal anti-Amb a 6 antibodies [32]. A significant association between IgE antibody responsiveness to Amb a 6 and the possession of HLA-DR5 was found in a genetic-epidemiologic study.…”

Expression and Self-Assembly of Norwalk Virus Capsid Protein from Venezuelan Equine Encephalitis Virus Replicons

“…4 and 5A) (26). To determine if the slight mobility shift was due to protein structure or alternative posttranslational processing, gradient-purified capsid proteins were extensively denatured with 5 M guanidine, reduced with dithiothreitol, and then alkylated with 0.25 M iodoacetic acid (23). Following NV1 sodium dodecyl sulfate-polyacrylamide gel electrophoresis FIG.…”

“…To further examine whether the BAC-Nor and VEE-Nor capsid proteins were indistinguishable, gradient-purified proteins were treated as described in the legend to Fig. 5, subjected to exhaustive trypsin digestion, and analyzed by high-pressure liquid chromatography and mass spectrometry at the Protein Core Facility at the University of North Carolina (23). rNV protease cleavage patterns were identical whether expressed from baculoviruses or from VEE (data not shown).…”

“…By Western blot analysis, VEE-Nor displayed a higher apparent molecular mass than that of BAC-Nor (rNV) (A). To remove potential conformational structure, VEE-Nor and BAC-Nor particles were further treated with 5 M guanidine-HCl at room temperature for 5 min to denature the proteins, the pH was adjusted to pH 8.6 with 4.6 M Tris, and the proteins were reduced with 0.1 M dithiothreitol (Sigma) for 45 min in the dark at room temperature before being alkylated by 0.25 M iodoacetic acid (Calbiochem) for 30 min at room temperature (23). The reduced and alkylated capsids were then diluted and analyzed by SDS-PAGE (B).…”