title: Expression of the recombinant major allergen of Salsola kali pollen (Sal k 1) and comparison with its low-immunoglobulin E-binding mutant.
abstract: BACKGROUND
  The inhalation of Salsola kali pollen is an important cause of pollinosis during summer and early fall throughout desert and semi-desert areas. Sal k 1 has been previously reported as a major allergen of S. kali pollen. In this study, we produced the recombinant Sal k 1 and also its low IgE-binding mutant form. We further compared the IgE binding ability of these two recombinant molecules.
  
  
  METHODS
  The recombinant Sal k 1 and its low IgE-binding variant, obtained by three amino acid exchanges (R(142)-->S, P(143)-->A, D(144)-->V), were cloned and expressed in E. coli, as proteins fused with thioredoxin and His-tags, and then purified by Ni2+ affinity chromatography. The IgE-binding capacity of the wild-type and mutated rSal k 1 was compared using immunoblotting, ELISA and inhibition assays by ten sera from S. kali allergic patients. Moreover, in vivo IgE-reactivity was investigated by the skin prick test.
  
  
  RESULTS
  Both the recombinant and the mutated form of Sal k 1 were expressed in E. coli at a relatively high amount and soluble form. All sera recognized rSal k 1 via immunoassay analysis. In addition, inhibition assays demonstrated that the purified rSal k 1 was similar to its counterpart in the crude extract. The mutated rSal k 1 exhibited a reduced IgE-binding capacity against wild-type rSal k 1.
  
  
  CONCLUSIONS
  This study demonstrates that purified rSal k 1 is comprised of IgE-epitopes similar to that of its natural counterpart and that the mutated variant showed a reduced IgE-binding capacity based on in vitro assays and in vivo provocation testing.
authors.affiliation: Immunology Department, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Mashhad, Iran.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Assarehzadegan
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Sankian
authors.forename: Farahzad
authors.initials: F
authors.lastname: Jabbari
authors.forename: Mohsen
authors.initials: M
authors.lastname: Tehrani
authors.forename: AbdolReza
authors.initials: A
authors.lastname: Varasteh
mesh_terms: D000328:Adult; D052179:Antigens, Plant / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D008297:Male; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011058:Pollen; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006255:Rhinitis, Allergic, Seasonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D031248:Salsola
chemical_list: D052179:Antigens, Plant; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E