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Published on: December 29, 2015
Non-structural protein 1 from Japanese encephalitis virus expressed in E. coli retains its molecular weight and
Jae-Won Choi1, Hyo-Ji Eom2, Hak Yong Kim1
1Department of Biochemistry, Chungbuk National University, Cheongju, 28644, Republic of Korea; School of Biological Sciences, Chungbuk National University, Cheongju, 28644, Republic of Korea.
Abstract:
Japanese encephalitis virus (JEV) is a member of the Flavivirus genus and has recently attracted attention as a high-risk pathogen in the Asia-Pacific region, with up to 30% mortality in the afflicted patients. Recent outbreaks of flavivirus-associated infections around the world have put the focus on non-structural protein 1 (NS1) as a candidate for diagnostic and vaccine researches on flaviviruses. Although the JEV NS1 protein has been expressed in eukaryotic cells, attempts to express JEV NS1 in E. coli are on due to advantages such as rapid growth, easy manipulation, low cost, and high yield. However, the challenges of low yield and poor solubility of the proteins expressed in E. coli remain to be overcome. Herein, we reported successful expression of the JEV NS1 protein in E. coli Rosetta(DE3) strain. We standardized the temperature, induction time, as well as the concentration of the inducer for optimizing the expression of JEV NS1 in E. coli. Further, we successfully obtained soluble JEV NS1 from inclusion bodies by partial refolding during elution and gradual refolding during dialysis. Furthermore, the JEV NS1 protein was found to retain its molecular weight and was able to induce an immune response in the mouse. Western blot and indirect enzyme-linked immunosorbent assay were performed using the blood of the immunized mouse and purified JEV NS1 in this study. Hence, JEV NS1 expressed in and isolated from E. coli Rosetta(DE3) strain holds potential for application in vaccine development and diagnostic studies to combat Japanese encephalitis outbreaks in the future.
Insights
Researchers successfully expressed Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) in E. coli, overcoming solubility issues. This soluble JEV NS1 protein shows potential for developing new vaccines and diagnostics against Japanese encephalitis.
Area of Science:
- Virology
- Molecular Biology
- Biotechnology
Background:
- Japanese encephalitis virus (JEV) is a significant flavivirus threat in the Asia-Pacific, causing severe illness with high mortality.
- The non-structural protein 1 (NS1) of flaviviruses is a key target for diagnostic and vaccine research.
- Expressing JEV NS1 in E. coli offers advantages but faces challenges in yield and solubility.
Purpose of the Study:
- To achieve successful expression of soluble Japanese encephalitis virus non-structural protein 1 (JEV NS1) in E. coli.
- To optimize expression conditions for JEV NS1 in E. coli.
- To demonstrate the potential of E. coli-expressed JEV NS1 for vaccine and diagnostic applications.
Main Methods:
- Optimized expression of JEV NS1 in E. coli Rosetta(DE3) by standardizing temperature, induction time, and inducer concentration.
- Developed a refolding strategy involving partial refolding during elution and gradual refolding during dialysis to obtain soluble NS1 from inclusion bodies.
- Validated the immunogenicity of the purified JEV NS1 protein in mice using Western blot and indirect ELISA.
Main Results:
- Successfully expressed JEV NS1 protein in E. coli Rosetta(DE3) strain.
- Obtained soluble JEV NS1 protein through a novel refolding process from inclusion bodies.
- Confirmed that the purified JEV NS1 retained its molecular weight and elicited an immune response in mice.
Conclusions:
- The developed method enables efficient expression and refolding of soluble JEV NS1 in E. coli.
- The E. coli-derived JEV NS1 protein is immunogenic and suitable for further development.
- This approach holds significant potential for advancing Japanese encephalitis diagnostics and vaccine development.
