Ni nta column

The ZIKV MTase (residues 1–266) coding fragment was inserted to the BamHI and XholI restriction sites of the pET28b-SUMO vector for expression in the Escherichia coli BL21 (DE3). Cells were grown in LB medium at 37°C and then induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside at 16°C. The cells were harvested and resuspended in lysis buffer A (25 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol and 2 mM β-mercaptoethanol) and lysed by high pressure homogenization. The supernatant after centrifugation was loaded onto the Ni-NTA column (GE). The column was washed using buffer A supplemented with 20 mM imidazole and eluted using buffer A supplemented with 250 mM imidazole. After concentration by ultrafiltration, protein sample was diluted into buffer B (50 mM HEPES, pH7.5, 0.5 M NaCl, 5% glycerol), and the fraction containing (His)₆-SUMO-ZIKV MTase was cleaved with ULP protease at 4°C overnight. The protein sample was then re-loaded on the Ni-NTA column to remove the (His)₆-SUMO and the ULP protease and further purified by sequential chromatography: cation exchange column (Hi Trap SP 5 ml, GE) and gel-filtration column (Superdex 75 10/300 GL, GE). MTase was finally concentrated to 10 mg/ml (0.33 mM) and mixed with 1.98 mM SAH prior to crystallization.

The expression process of Ub and Lb^pro was the same as that of SARS-CoV-2 PL^pro. Both Ub and Lb^pro were first purified by a Ni-NTA column (GE Healthcare), followed by buffer exchange using a Superdex75 (GE Healthcare) pre-equilibrated with a buffer containing 50 mM HEPES, 150 mM NaCl, pH 8.0. The fractions were flash-frozen in liquid nitrogen and stored at −80 °C for further use. Ub-AMC was synthesized according to previous references [31 (link)]. Specifically, Gly-Gly-AMC was dissolved in DMSO to a saturated solution and mixed with an equal volume of Ub stock solution (5 mM). After that, Lb^pro (40 μM) was added to the reaction solution at room temperature. After incubating for about 6 h, the reaction mixture was purified by a Ni-NTA column (GE Healthcare). The flow-through was further purified by Superdex 75 (GE Healthcare) pre-equilibrated with a buffer containing 50 mM HEPES, 150 mM NaCl, and pH 8.0. The fraction of the second main peak in the chromatogram was collected and stored at −80 °C for further use.

Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), Acros (New Jersey, NJ, USA), Alfa-Aesar (Ward Hill, MA, USA), Ambeed (Arlington Heights, IL, USA), or AK Scientific (Union City, CA, USA) and were of reagent grade or better. The PD-10 column and Ni-NTA columns used for protein purification were purchased from GE Healthcare (Piscataway, NJ, USA).