*2.13. TMD2-M*/*E Expression*

To express recombinant E proteins from ZIKV in mammalian cells, TMD2-prM (TransMembrane Domain II) and E genes from MR766 and BR15, as well as a mutant BR15 bearing residues E-152 to E-158 from MR766, were synthesised by GeneCust (Boynes, France). Recombinant proteins comprised aa 275 to aa 775 of polyproteins, which correspond to the very end of prM protein (TMD2, used as signal peptide for E proteins) and the entire E protein. As Flavivirus prM protein plays a role of chaperone to ensure the proper folding of the E protein, we expected that viral chaperone activity eviction would have exacerbate differences in E protein folding [34]. Modifications to optimize viral E protein expression in human cells were done on original protein sequences. Then, mammalian codon-optimised sequences coding for TMD2-prM and E proteins were cloned into the *NheI* and *NotI* restriction sites of the pcDNA3.1(-) plasmid to generate pMR766, pBR15 and pBR15E-MUT, respectively. Each plasmid was transfected in human HEK-293T cells using lipofectamine 3000 according to manufacturer's instructions.
