Unique properties of Zika NS2B-NS3pro complexes as decoded by experiments and MD simulations

Amrita Roy; Liangzhong Lim; Shagun Srivastava; Jianxing Song

Cloning, expression and purification of linked and unlinked Zika NS2B-NS3pro-Based on the sequence alignment with NS2B and NS3pro of the Dengue serotype 2 we previously characterized (12) , the corresponding Zika sequences were identified for the NS2B (Fig. 1A) and NS3pro (Fig. 1B) of the Asian Zika virus. From synthetic genes with E. coli preferred codons, we amplified and subsequently cloned the DNA fragments into His-tagged expression vectors, which encode the isolated NS2B (48-100) with the transmembrane regions deleted (Fig. 1A) ; as well as isolated NS3 (14-185) (Fig. 1B) . The expression level of the recombinant NS2B was extremely low, and consequently we needed to grow many liters of E. coli cells to obtain sufficient NS2B for refolding with NS3pro by the same protocol we previously utilized for the Dengue one (12) . The refolded NS2B-NS3pro with His-tag (column 2 of Fig. 1C ) was cleaved by thrombin covalently linked to beads to remove the His-tag, followed by binding to an excess amount of the Ni 2+ -beads to remove His-tag and uncleaved fusion protein, as well as final purification with FPLC gel-filtration chromatography to obtain unlinked NS2B-NS3pro without His-tag (column 3 of Fig. 1C ). We attempted to refold Zika NS3pro without NS2B and found that NS3pro completely precipitated during refolding, thus indicating that Zika NS3pro also absolutely requests its NS2B cofactor for the correct folding, as previously observed on other flavivirus NS3 proteases (12) (13) (14) (15) (16) (17) (18) .