High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: a major allergen of shrimp.
Well-characterised and immunologically active recombinant allergens are of eminent importance for improvement of diagnostic tools and immunotherapy of allergic diseases. The use of recombinant allergens has several advantages such as the more precise quantification of the active substance compared to allergen extracts and the reduced risk of contamination with other allergenic proteins compared to purified natural allergens. Optimised standard protocols for expression and purification and a detailed physico-chemical characterisation of such recombinant allergens are necessary to ensure consistent quality and comparability of results obtained with recombinant material. In this study the major allergen Pen a 1 of brown shrimp (Penaeus aztecus) was expressed in E. coli and purified in two steps by immobilised metal chelate-affinity chromatography (IMAC) and size-exclusion chromatography. Identity and purity were verified with N-terminal sequencing and peptide mass fingerprinting. Circular dichroism and NMR-spectroscopy indicated an alpha-helical flexible structure of rPen a 1 which is in accordance with the known structure of tropomyosins. Finally, the recombinant allergen proved to be immunologically reactive in IgE Western blot analysis and ELISA. This study provides a protocol for the preparation of recombinant shrimp tropomyosin in standardised quality.
Albrecht
Melanie
M
Department of Allergology, Paul-Ehrlich-Institut, Langen, Germany.
Alessandri
Stefano
S
Conti
Amedeo
A
Reuter
Andreas
A
Lauer
Iris
I
Vieths
Stefan
S
Reese
Gerald
G
D000485:Allergens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D006801:Humans; D007120:Immunochemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification
D000485:Allergens; C513564:Pen a 1 allergen, Penaeus aztecus; D011994:Recombinant Proteins