Volume 4, Issue 1 (Vol.4 No.1 Oct 2015)                   rbmb.net 2015, 4(1): 19-24 | Back to browse issues page

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Moghadam M, Ganji A, Varasteh A, Falak R, Sankian M. Refolding Process of Cysteine-Rich Proteins: Chitinase as a Model . rbmb.net. 2015; 4 (1) :19-24
URL: http://rbmb.net/article-1-67-en.html
Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran
Abstract:   (2374 Views)

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 32 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.

Full-Text [PDF 375 kb]   (609 Downloads)    
Subject: Biochemistry
Received: 2016/08/22 | Accepted: 2016/08/22 | Published: 2016/08/22

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