Expression of the recombinant major allergen of Salsola kali pollen (Sal k 1) and comparison with its low-immunoglobulin E-binding mutant.
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.
Assarehzadegan
Mohammad Ali
MA
Immunology Department, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Mashhad, Iran.
Sankian
Mojtaba
M
Jabbari
Farahzad
F
Tehrani
Mohsen
M
Varasteh
AbdolReza
A
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
D052179:Antigens, Plant; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E