Solubilization and Refolding of Inclusion Body of Grapevine fanleaf virus-coat Protein Produced in E. coli

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Article Type:
Research/Original Article (دارای رتبه معتبر)
Abstract:
Objective
 High level expression of recombinant protein in Escherichia coli often results in aggregation of the expressed protein molecules into inclusion bodies. Cysteines in the protein contribute to this process. Intermolecular and intramolecular disulfide bonds formation in Grapevine fanleaf virus (GFLV)-coat protein (CP), a cysteine-rich protein, and lead to aggregation when the recombinant protein was overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.  
Materials and methods
In this paper, SDS/MPD method used as a unique approach to refold the structure some protein in the presence of the denaturing agent, Sodium Dodecyl Sulfate (SDS). This was made possible by addition of the amphipathic solvent 2,4-Methyl-2-PentaneDiol (MPD), used as protecting but also as refolding agent for these proteins. For this purpose, the inclusion bodies containing insoluble proteins of GFLV CP were solubilized by denaturing with SDS, then the soluble proteins were purified by the size exclusion chromatography, and finally, the purified proteins were refolded using SDS/MPD method.  
Results
Transmission electron microscopy images confirmed the reassembly GFLV VLPs using SDS/MPD method.  
Conclusions
MPD modulate the denaturing properties of SDS, therefore, for the first time, a simple and effective method to refolded GFLV VLP from the SDS-denatured state.
Language:
Persian
Published:
Journal of Agricultural Biotechnology, Volume:11 Issue: 1, 2019
Pages:
151 to 167
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