*2.1. Expression and Purification of MTase*

For studying the biochemical and biophysical characteristics of the enzyme MTase, the gene for MTase was cloned into the pET-28a vector and validated by restriction digestion of a ~969 bp fragment (Figure 1A) and confirmed by DNA sequencing (data not shown). The recombinant MTase construct was transformed into BL21-DE3 cells to express the protein, as demonstrated using SDS-PAGE and Western blotting with proper controls (Figure 1B,C; Lanes 2 and 3). The protein was solubilized using different detergents, but achieved maximum solubility with 0.5% NLS (Figure 1B,C; Lane 4). The recombinant protein of 37 kDa, with a His-tag, was purified using Ni-NTA chromatography and confirmed to be MTase, as shown by SDS-PAGE and western blotting using epitope-specific antibodies (Figure 1B,C; Lane 5). In conclusion, the protein was purified and confirmed to be MTase through enzyme assay using GTP as the substrate.

**Figure 1. Cloning, expression, and purification of MTase:** (**A**) The cloned MTase gene (969 bp) was confirmed by restriction digestion using NdeI and XhoI (Panel A). (**B**) Coomassie-stained gel representing the expressed and purified fractions of HEV MTase. Lane 1, marker; lane 2, uninduced cell lysate; lane 3, induced cell lysate; lane 4, solubilised fraction; lane 5, Ni-NTA purified fraction. (**C**). Western blotting analysis using anti-HEV MTase primary antibody. Lane 1, marker; lane 2, uninduced cell lysate; lane 3, induced cell lysate; lane 4, solubilised fraction; lane 5, Ni-NTA purified fraction. **Figure 1. Cloning, expression, and purification of MTase:** (**A**) The cloned MTase gene (969 bp) was confirmed by restriction digestion using NdeI and XhoI (Panel A). (**B**) Coomassie-stained gel representing the expressed and purified fractions of HEV MTase. Lane 1, marker; lane 2, uninduced cell lysate; lane 3, induced cell lysate; lane 4, solubilised fraction; lane 5, Ni-NTA purified fraction. (**C**) Western blotting analysis using anti-HEV MTase primary antibody. Lane 1, marker; lane 2, uninduced cell lysate; lane 3, induced cell lysate; lane 4, solubilised fraction; lane 5, Ni-NTA purified fraction.
