Expression and characterization of three important panallergens from hazelnut.
Several hazelnut allergens with different clinical relevance and crossreactive properties have been identified and characterized so far. The aim of this study was to develop protocols for producing relatively large amounts of three recombinant hazelnut allergens Cor a 1.04, Cor a 2, and Cor a 8 in a folded and immunologically active form. The availability of well-characterized, pure recombinant allergens will improve diagnostic in vitro tests for food allergy, by allowing a highly sensitive component resolved diagnosis. Depending on the individual hazelnut allergen, protocols for heterologous production - either as fusion or nonfusion protein - were developed to obtain homogenous protein batches. The resulting proteins were purified by a two-step FPLC method and their IgE antibody reactivity was verified. Identity was verified by N-terminal sequencing and MALDI-TOF-MS analysis. Their secondary and tertiary structure was controlled by circular dichroism (CD)-spectroscopy and NMR analysis. Decisions on the strategies for expression and purification of allergens on a large scale were made on a case by case basis: Preparation of rCor a 1.04 and rCor a 2 as fusion proteins in E. coli from inclusion bodies resulted in approximately 10 mg pure protein per liter whereas rCor a 8 expression in yeast as nonfusion protein yielded 30 mg/L.
Lauer
Iris
I
Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany. lauir@pei.de
Alessandri
Stefano
S
Pokoj
Sven
S
Reuter
Andreas
A
Conti
Amedeo
A
Vieths
Stefan
S
Scheurer
Stephan
S
D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D031211:Corylus / Q000276:immunology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D021184:Nut Hypersensitivity / Q000209:etiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification
D000485:Allergens; D011994:Recombinant Proteins