Structural and Immunological Characterization of Recombinant Pan b 1, a Major Allergen of Northern Shrimp, <b><i>Pandalus borealis</i></b>

Abstract

Background: Shellfish allergy is one of the major causes of life-threatening allergic reactions to food. The shrimp species Pandalus borealis is the commercially most important coldwater shrimp species, and its protein extract is commonly used in shrimp allergy diagnostics. However, the DNA sequence of its major allergen, tropomyosin, designated Pan b 1, was not previously described. Our aim was to identify the cDNA sequence of Pan b 1 and to generate a recombinant protein with similar structure and allergenicity as the natural protein. Methods:P. borealis shrimps were caught in the Oslofjord (Norway). cDNA from Pan b 1 was generated, an N-terminal histidine tag was added, and the protein was expressed in Escherichia coli. The recombinant Pan b 1 was characterized by structural and IgE-binding studies and investigated further with basophil activation tests (BATs) and skin prick tests (SPTs) on Norwegian shrimp-allergic individuals. Results: The open reading frame encoded 284 amino acids that shared 97–100% identity with other shrimp tropomyosins. Mass spectroscopy of natural Pan b 1 confirmed the protein’s molecular mass and indicated the absence of posttranslational modifications. Circular dichroism spectroscopy revealed virtually identical spectra between recombinant and natural Pan b 1, which together with native PAGE and size exclusion chromatography results indicated a similar structure. Furthermore, immunoblot and ELISA studies as well as BATs and SPTs showed equivalent results of recombinant and natural Pan b 1. Conclusion: A recombinant tropomyosin from P. borealis was generated that can be used in diagnostics and further studies on tropomyosin allergenicity and specific immunotherapy.

Mapping of the Immunodominant Regions of Shrimp Tropomyosin Pan b 1 by Human IgE-Binding and IgE Receptor Crosslinking Studies

“…IgE-binding regions earlier identified for F. aztecus tropomyosin (Pen a 1) [20] are marked with shading. Pan b 1 differs from Pen a 1 in nine amino acids localized between amino acid number 44 and 72 [25]. T = Pan b 1.0101 (GenBank accession No.…”

“…It was previously shown by CD spectroscopy that nPan b 1 has a predominantly α-helical structure with the characteristic minima at 208 and 222 nm and maximum at 192 nm [25,30] and that the 22 amino acid His-tag added to the Pan b 1 fragments did not interfere with the folding of rPan b 1 [25]. CD spectroscopy of the Pan b 1 fragments in buffer with physiological salt content (PBS; fig.…”

“…A cDNA clone with Pan b 1.0101 was obtained as described in Myrset et al [25]. Five overlapping fragments of Pan b 1.0101 cDNA, spanning the entire open reading frame, were amplified and cloned into the vector pET19b (Novagen, Darmstadt, Germany) using In-Fusion cloning (Clontech, Saint-Germain-en-Laye, France) according to the manufacturer's instructions (F1-F5):…”

“…These patients were part of a previous related study including 6 patients [25], and the designation of the patients is therefore continued in the current study (patient A, B, C, E and F). For control experiments, 3 individuals with negative SPTs to shrimp extract were included for the basophil activation tests (BAT), and 3 non-atopic individuals were included for the SPTs.…”

“…For this, we generated five overlapping recombinant fragments spanning the entire sequence of tropomyosin from the Northern Atlantic shrimp species Pandalus borealis (Pan b 1). The amino acid sequence of Pan b 1 was recently identified by our group [25]. …”