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

• Published in: International Archives of Allergy and Immunology
• Main Authors: Myrset, Heidi R., Barletta, Bianca, Di Felice, Gabriella, Egaas, Eliann, Dooper, Maaike M.B.W.
• Format: Article in Journal/Newspaper
• Language: English
• Published: S. Karger AG 2012
• Subjects: Norway; northern shrimp; Pandalus borealis
• Online Access: http://dx.doi.org/10.1159/000339740; https://www.karger.com/Article/Pdf/339740

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.