Structural and immunological characterization of recombinant Pan b 1, a major allergen of northern shrimp, Pandalus borealis.
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.


CONCLUSIONS
A recombinant tropomyosin from P. borealis was generated that can be used in diagnostics and further studies on tropomyosin allergenicity and specific immunotherapy.
Norwegian Veterinary Institute, Oslo, Norway. heidi.myrset@gmail.com
Heidi R
HR
Myrset
Bianca
B
Barletta
Gabriella
G
Di Felice
Eliann
E
Egaas
Maaike M B W
MM
Dooper
D000328:Adult; D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000276:immunology*; D015549:Basophil Degranulation Test; D002478:Cells, Cultured; D019143:Evolution, Molecular; D005260:Female; D005512:Food Hypersensitivity / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D033542:Pandalidae / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology*; D012758:Shellfish / Q000009:adverse effects; D014335:Tropomyosin / Q000276:immunology* / Q000378:metabolism
D000485:Allergens; D060829:Arthropod Proteins; C579817:Pan b 1 allergen, Pandalus borealis; D011994:Recombinant Proteins; D014335:Tropomyosin; D007073:Immunoglobulin E