Production of envelope protein domain III of Zika Virus in Escherichia coli: evaluation of the protein obtained from soluble fraction and inclusion bodies

Abstract

Zika Vírus (ZIKV) is an arbovirus that belongs to the Flaviviridae family and Flavivirus genus. It is capable of infecting humans and causing neurological complications, including Guillain-Barré Syndrome and Microcephaly. The main function of ZIKV envelope E protein is the interaction with cell receptors to promote the fusion between viral envelope and host cell membrane. The E protein is composed of three domains (I, II and III). The domain III (EDIII) is the region that interacts with the cell receptor, which makes it an important target for production of neutralizing antibodies that would inhibit the viral adsorption. Different studies have shown the importance of the EDIII in viral neutralization and its potential for the development of vaccines against ZIKV, in addition to its potential, it can be used as discriminant antigen to be implemented in serological tests for the diagnosis of Zika. During the student's master degree period, the HisEDIIIHis production process was developed. However, the purification process of this protein was impracticable. As an alternative process, two other gene constructions were synthesized; HisEDIII, with only one His-tag and removal of hydrophobic portions, and Fh8EDIII, which was fused with Fh8 solubility tag. The results of the production of these two new proteins were satisfactory, but there is further potential for production optimization. For the PhD, the proposal is to compare three technological routes: HisEDIII obtained from the soluble fraction, HisEDIII obtained from the inclusion bodies and Fh8EDIII obtained from the soluble fraction. We also expect to improve the production, purification, and up-scale to the bioreactor using the most viable strategy that would allow obtaining the EDIII with preserved antigenicity and functionality. It is expected that the results from this study will contribute to wide discussion within the scientific community regarding the most optimal strategy for production and purification of proteins that have low solubility and physicochemical characteristics similar to EDIII. In addition, this work shows academic originality and an utmost importance in application, as there is a need, which is not yet achieved, of economically accessible and specific serological tests, and ultimately for effective vaccines against the Zika Virus. (AU)