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Published on: April 23, 2012
Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in
Maha Masoudi1, Ali Teimoori2, Alijan Tabaraei3
1Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
Insights
We optimized rotavirus VP6 protein expression in E. coli using sequence and codon adaptation. This novel method significantly increased recombinant VP6 (rVP6) yield, paving the way for cheaper diagnostic kits and vaccines.
Area of Science:
- Virology
- Molecular Biology
- Biotechnology
Background:
- Rotavirus is a major cause of infectious diarrhea in young children.
- Rotaviruses are classified into serogroups (A-J) based on the VP6 protein.
- Efficient production of VP6 is crucial for diagnostics and vaccine development.
Purpose of the Study:
- To design and implement a novel VP6 sequence optimization strategy.
- To enhance the expression level of the rotavirus VP6 protein in E. coli.
- To establish an efficient method for producing high-yield recombinant VP6 (rVP6).
Main Methods:
- Sequence optimization including codon adaptation index, codon pair bias, and GC content.
- Redesign of the ribosome binding site (RBS) and optimization of VP6 mRNA secondary structure.
- Analysis of expression conditions (IPTG concentration, temperature, induction time) and purification of rVP6.
Main Results:
- Achieved high expression of rVP6 (43.8 g/L) in insoluble aggregates.
- Purified rVP6 yield was 10.83 g/L.
- Optimized conditions were 37°C, 1 mM IPTG, and 8-hour induction.
Conclusions:
- Novel sequence optimization significantly increased rVP6 production compared to previous reports.
- The optimized rVP6 can be used to generate specific antiserum for diagnostics.
- This approach holds potential for developing cost-effective rotavirus diagnostic kits and vaccines.
Abstract:
Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.
