Refolding Process of Cysteine-Rich Proteins: Chitinase as a Model
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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Varastegan Institute for Medical Sciences
2015-10-01
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| Series: | Reports of Biochemistry and Molecular Biology |
| Subjects: | |
| Online Access: | http://rbmb.net/article-1-67-en.pdf |
| Summary: | 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. |
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| ISSN: | 2322-3480 2322-3480 |