Expression of the recombinant major allergen of salsola kali pollen (Sal k 1) and comparison with its low-immunoglobulin E-binding mutant

Assarehzadegan, M. A. and Sankian, M. and Jabbari, F. and Tehrani, M. and Varasteh, A. (2010) Expression of the recombinant major allergen of salsola kali pollen (Sal k 1) and comparison with its low-immunoglobulin E-binding mutant. Allergology International, 59 (2). pp. 213-222.

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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 (R142→S, P143→A, D144→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. © 2010 Japanese Society of Allergology.

Item Type: Article
Additional Information: Cited By :12 Export Date: 16 February 2020 CODEN: ALINF Correspondence Address: Varasteh, A.; Immunology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran; email: varasteha@mums.ac.ir
Uncontrolled Keywords: Comparison Expression Mutant Sal k 1 Salsola kali histidine immunoglobulin E recombinant allergen recombinant protein Sal k 1 thioredoxin unclassified drug hybrid protein plant antigen protein binding adult affinity chromatography amino acid substitution article clinical article controlled study enzyme linked immunosorbent assay Escherichia coli female human immunoassay immunoblotting male nucleotide sequence pollen pollen allergy priority journal provocation test Salsola genetics hay fever immunology metabolism molecular cloning mutation site directed mutagenesis Antigens, Plant Chromatography, Affinity Cloning, Molecular Humans Mutagenesis, Site-Directed Recombinant Fusion Proteins Rhinitis, Allergic, Seasonal
Subjects: WH Hemic and Lymphatic System
Divisions: Mashhad University of Medical Sciences
Depositing User: mr lib4 lib4
Date Deposited: 03 Mar 2020 06:35
Last Modified: 03 Mar 2020 06:35
URI: http://eprints.mums.ac.ir/id/eprint/12928

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