The ED3 sequences of DENV1, DENV2, DENV3, and DENV4 serotypes were retrieved from the UniProt database, and the nucleotide sequences, optimized for expression in Escherichia coli, were synthesized and cloned at the NdeI and BamHI sites of pET15b (Novagen) [26 (link)].
All ED3 variants were overexpressed in E. coli JM109(DE3)pLysS as inclusion bodies and refolded as described previously [42 (link)]. In short, after harvesting, the cells were lysed in lysis buffer (150 mM NaCl, 0.5% sodium deoxycholate, and 1% SDS in 50 mM Tris-HCl pH 8.5) and lysis wash buffer (lysis buffer supplemented with 1% v/v NP-40) through sonication. The cell lysates were air oxidized for 36 h at 30 °C in 6 M guanidine hydrochloride in 50 mM Tris-HCl, pH 8.7. The His6-tagged ED3s were purified by Ni-NTA (Wako, Tokyo, Japan) chromatography, followed by dialysis against 10 mM Tris-HCl, pH 8.0 at 4 °C. The N-terminal His6-tag was cleaved by thrombin proteolysis [43 (link),44 (link)]. ED3s were purified by a second round of Ni-NTA chromatography followed by reversed-phase (RP) HPLC. The proteins were lyophilized and stocked as powder at −40 °C until use [21 (link)].
All ED3 variants were overexpressed in E. coli JM109(DE3)pLysS as inclusion bodies and refolded as described previously [42 (link)]. In short, after harvesting, the cells were lysed in lysis buffer (150 mM NaCl, 0.5% sodium deoxycholate, and 1% SDS in 50 mM Tris-HCl pH 8.5) and lysis wash buffer (lysis buffer supplemented with 1% v/v NP-40) through sonication. The cell lysates were air oxidized for 36 h at 30 °C in 6 M guanidine hydrochloride in 50 mM Tris-HCl, pH 8.7. The His6-tagged ED3s were purified by Ni-NTA (Wako, Tokyo, Japan) chromatography, followed by dialysis against 10 mM Tris-HCl, pH 8.0 at 4 °C. The N-terminal His6-tag was cleaved by thrombin proteolysis [43 (link),44 (link)]. ED3s were purified by a second round of Ni-NTA chromatography followed by reversed-phase (RP) HPLC. The proteins were lyophilized and stocked as powder at −40 °C until use [21 (link)].
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