Refolding Process of Cysteine-Rich Proteins: Chitinase as a Model

Publish Year: 1394
نوع سند: مقاله ژورنالی
زبان: English
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شناسه ملی سند علمی:

JR_RBMB-4-1_003

تاریخ نمایه سازی: 10 شهریور 1400

Abstract:

Background: Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins. Methods: Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously. Results: After protein solubilization and refolding, SDS-PAGE showed a ۳۲ kDa band that was recognized by an anti-chitin antibody on western blots. Conclusion: By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.

Authors

Malihe Moghadam

Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran

Ali Ganji

Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran

Abdolreza Varasteh

Allergy Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran

Reza Falak

Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran - Department of Immunology, Iran University of Medical Sciences, Tehran, Iran

Mojtaba Sankian

Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran