Purification and immunobiochemical characterization of folding variants of the recombinant major wasp allergen Ves v 5 (antigen 5).
BACKGROUND
Antigen 5 is one of three major allergens in wasp venoms, but unlike phospholipase A(1) and hyaluronidase, both of which are enzymes, its biological function is unknown. The cDNA coding for this allergen has been isolated and used for recombinant expression. Thorough analysis of the expression product is essential in order to evaluate the usefulness for in vivo or in vitro application.


OBJECTIVE
In this study, folding variants of the recombinant major allergen Ves v 5 from Vespula vulgaris were immunologically and biochemically investigated in order to determine their possible applicability for diagnostic or therapeutic purposes.


METHODS
The cDNA encoding Ves v 5 was cloned into the expression vector pSE420 which generates recombinant products lacking a tag sequence. After expression, inclusion bodies were purified, subsequently denatured and dialyzed against different solutions. The structural properties of soluble proteins were analyzed by size exclusion chromatography, non-reducing SDS-PAGE, native PAGE, N-terminal sequencing, proteolytic digestion and ion exchange chromatography. Immunological investigations were performed by using different monoclonal antibodies (mAbs) specific for Ves v 5 and IgE from patients allergic to wasp venom allergens.


RESULTS
After dialysis, soluble monomeric recombinant Ves v 5 was more than 95% pure in each case. Using different dialysis solutions, clearly distinguishable folding variants were obtained. In one case, the recombinant allergen was comparable with the natural counterpart in respect of migration in non-reducing SDS-PAGE, native PAGE and IgE reactivity. This variant reacted with two different Ves v 5-specific mAbs and produced a stable fragment after proteolytic digestion. Elution from a cation exchange chromatography column was achieved with 320 mM NaCl. In two other cases, folding variants exhibited a different migration behavior in SDS-PAGE and native PAGE compared with the natural allergen. Also, the mAb 1E11 recognized none of these variants since it presumably detected a conformational epitope. Moreover, the IgE reactivity was clearly reduced and proteolytic digestion effected almost complete degradation. These variants eluted from the cation exchange column with 400 mM NaCl.


CONCLUSIONS
Defined folding strategies resulted in both soluble misfolded variants with reduced IgE reactivity, potentially suitable for immunotherapy, and natural-like folded variants for diagnosis.
Suck
R
R
Allergopharma Joachim Ganzer KG, Reinbek, Germany.
Weber
B
B
Kahlert
H
H
Hagen
S
S
Cromwell
O
O
Fiebig
H
H
D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014862:Wasp Venoms / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D000485:Allergens; D011994:Recombinant Proteins; D014862:Wasp Venoms; C040583:white-faced hornet venom antigen 5