title: A folded and immunogenic IgE-hyporeactive variant of the major allergen Phl p 1 produced in Escherichia coli.
abstract: BACKGROUND
  Group 1 grass pollen allergens are a major cause of allergic disease. Specific immunotherapy involving controlled administration of allergens can be used as a disease-modifying treatment for such disease. Recombinant allergen variants with reduced IgE binding capacity may be used as component in such vaccines, as they may induce fewer treatment side effects than materials currently in use. A mutated variant of the immunodominant C-terminal domain of the group 1 grass pollen allergen Phl p 1 was recently established through an approach that used a set of human monoclonal IgE as a guide to identify mutations that disturbed IgE-allergen interactions. Further analysis of this domain is required to establish its potential for use in treatment.
  
  
  METHODS
  GST-tagged wild-type and mutated C-terminal domains of Phl p 1 were produced in Escherichia coli TUNER(DE3). The products were purified by affinity chromatography on immobilized glutathione. GST was removed by enzymatic cleavage and tag-free products were purified by size exclusion chromatography. Products were assessed by SDS-PAGE, circular dichroism spectroscopy, differential scanning fluorimetry and dynamic light scattering. Rats were immunized with GST-tagged and tag-free mutated C-terminal domain of Phl p 1. Antigen-binding properties of induced antibodies were assessed by immunochemical analysis.
  
  
  RESULTS
  The mutated domain has a structure very similar to that of the wild-type domain as determined by circular dichroism, but a reduced thermal stability. Immunization of rats demonstrates that this IgE-hyporeactive domain, despite its three sequence modifications (K8A, N11A, D55A), is able to induce antibodies that substantially block the binding of allergic subjects' IgE to the wild-type allergen.
  
  
  CONCLUSIONS
  It is concluded that this IgE-hyporeactive molecule can be produced in folded form and that it is able to induce an antibody response that efficiently competes with IgE recognition of Phl p 1. These findings suggest that it, or a further evolved variant thereof, is a candidate for use as a component in specific immunotherapy against grass pollen allergy.
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village building 406, S-223 81, Lund, Sweden. mattias.levin@immun.lth.se.
authors.forename: Mattias
authors.initials: M
authors.lastname: Levin
authors.affiliation: Crystallization facility at the MAX IV laboratory and Lund University, Lund, Sweden. harm.otten@maxlab.lu.se.|Lund Protein Production Platform (LP3), Lund University, Lund, Sweden. harm.otten@maxlab.lu.se.
authors.forename: Harm
authors.initials: H
authors.lastname: Otten
authors.affiliation: Lund Protein Production Platform (LP3), Lund University, Lund, Sweden. claes.von_wachenfeldt@biol.lu.se.
authors.forename: Claes
authors.initials: C
authors.lastname: von Wachenfeldt
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village building 406, S-223 81, Lund, Sweden. mats.ohlin@immun.lth.se.
authors.forename: Mats
authors.initials: M
authors.lastname: Ohlin
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007114:Immunization; D007073:Immunoglobulin E / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011058:Pollen / Q000276:immunology*; D011485:Protein Binding; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000485:Allergens; D011994:Recombinant Proteins; D007073:Immunoglobulin E