Enhancing Solubilization of Hydrophobic ORF3 Product of Hepatitis E Virus in Bacterial Expression System
Journal Title: Journal of Vaccine & Immunotechnology - Year 2014, Vol 1, Issue 1
Abstract
Hepatitis E Virus (HEV) is a major cause of acute clinical hepatitis in developing countries. HEV genome has three open reading frames: ORF1, ORF2 and ORF3. ORF3 encodes a small 13 kDa protein (hereinafter called vp13). This protein has been suggested to have multiple functions, including interference with cell signaling pathways, viral particle release from infected cells, and viral pathogenesis. However, the structural basis and mechanism by which the vp13 exerts these multiple biological functions is unclear. Characterization of vp13 has been hindered by the difficulty in efficiently producing enough hand pure vp13 proteins. This may attribute to vp13’s hydrophobic and insoluble nature. The objective of this study was to express vp13 in prokaryotic expression system and optimize the conditions to increase the soluble portion for vp13 purification. The ORF3 sequence was cloned into a bacterial expression plasmid with maltose binding protein (MBP) as a tag. The recombinant plasmid was transformed into E. coli BL-21 strain for protein expression. Most MBP-vp13 protein was found in insoluble portion, making protein purification a challenge. To increase the solubility of this recombinant protein, we optimized the induction and processing conditions. Induction at 18°C and addition of 1% glucose to the bacterium culture successfully led to efficient expression of soluble recombinant MBP-vp13 fusion protein. Further, the soluble fusion protein was easy to be purified. This enhanced solubilization and purification of vp13 protein should facilitate further study of vp13’s structure and function. This study provides an example for expression and purification of hydrophobic proteins for vaccine or immunological reagent production.
Authors and Affiliations
Yanjin Zhang
Keywords
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- EP ID EP206073
- DOI 10.13188/2377-6668.1000002
- Views 81
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