Purification, refolding, and pH-dependent stability evaluation of Zika virus EDIII protein

Zika virus (ZIKV) infection, primarily spread through mosquito bites, poses a significant threat as global temperatures continue to rise. While vector control remains the primary strategy for mitigation, the design and development of cost-effective vaccines are essential to prevent future outbreaks, particularly in low- and middle-income countries. In our study, we have produced recombinant ectodomain III (rEDIII) of ZIKV from the E. coli host expression system. Further, we optimized the effect of temperature and growth media on the expression of ZIKV rEDIII. Our study establishes a systematic protocol for extracting functional ZIKV rEDIII since the majority of the expressed protein goes into inclusion bodies (IBs). We have optimized the refolding process of rEDIII in buffers with varying pH levels and demonstrated the association between the buffer environment and the refolding efficiency of ZIKV rEDIII. Among these, Tris-HCl pH 8.8 buffer successfully restored the native conformation of ZIKV rEDIII, as confirmed by CD spectroscopy. We further confirmed the biological activity of refolded ZIKV rEDIII protein by inhibiting ZIKV entry into the host cells in an in vitro competitive viral inhibition assay. Our study proposes suitable conditions required for refolding bacterially expressed ZIKV rEDIII protein into its functionally active form with a median 50% inhibitory dose (ID50) value of 29 µg. The results of this investigation extend beyond the current study as the presented approach can be used to purify and refold other viral proteins expressed as IBs, thereby contributing to the advancement of diagnostic and therapeutic strategies for flavivirus infections.