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

Document Type : Research Paper

Authors

1 Plant Pathology Department, Faculty of Agriculture, Tarbiat Modares University, Teheran, Iran

2 Department of Biophysics, Faculty of Biological Sciences Tarbiat Modares University, Tehran, Iran

3 Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1,6708 PB, Wageningen, The Netherlands

4 Tehran-Iran

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.
 
 
 
Yazdani R, Arab SS, Hassani-Mehraban A, Shams-Bakhsh M (2019) Solubilization and refolding of inclusion body of Grapevine fanleaf virus-coat protein produced in E. coli. Agricultural Biotechnology Journal 11 (1), 151-167. 
 
Agricultural Biotechnology Journal 11 (1), 151-167.
DOI: 10.22103/jab.2019.13125.1090
Received:  January 24, 2019; Accepted: April 28, 2019
© Faculty of Agriculture, Shahid Bahonar University of Kerman-Iranian Biotechnology Society

Keywords

References

یزدانی راضیه (1397) بررسی امکان نمایش اپی توپ­های خارجی در سطح ویروس برگ بادبزنی مو. رساله دکتری ویروس شناسی گیاهی، دانشگاه تربیت مدرس، تهران، ایران.
 
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Agricultural Biotechnology Journal
Volume 11, Issue 1 - Serial Number 33
Volume 11, No 1
June 2019
Pages 151-167

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