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

Mojtaba Sankian, Malihe Moghadam, Ali Ganji, Abdolreza Varasteh, Reza Falak, Mojtaba Sankian
2015 Reports of Biochemistry & Molecular Biology   unpublished
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
more » ... ecombinant 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.
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