Advanced sequence optimization for the high efficient yield of human group A Rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Masoudi, Maha; Teimoori, Ali; Tabaraei, Alijan; Shahbazi, Majid; Divbandi, Marzieh; Lorestani, Nazanin; Yamchi, Ahad; Nikoo, Hadi Razavi

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into ten 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 GC content were adapted based on E. coli codon usage. In addition, the ribosome binding site of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 mRNA was optimized in the whole length of the coding sequence. Various factors including IPTG concentration, temperature and induction time were analyzed for the optimization of the best expression in E. coli by SDS-PAGE and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyper-immune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and post inductions, the level of rVP6 was higher at 37°C, 1mM of IPTG, and 8 hours, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g.L-1 ). After purification, the yield of rVP6 was 10.83 g.L-1 . The rVP6 specific antiserum was confirmed by both Immunofluorescent and western blot analyses. 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 in order to diagnose serological tests and new rotavirus vaccines. This article is protected by copyright. All rights reserved.