Overexpression and Purification of Glycine Methyltransferases

The TVD_RS00875 and TVD_RS00880 genes, coding for the putative glycine methylation pathway, were inserted into BamHI-digested pET-28a(+), respectively, using the T5 exonuclease-dependent assembly system (Xia et al., 2019 (link)). The obtained E. coli strains grown with 50 μg/mL kanamycin were used to overexpress TVD_RS00875 (TvGMT) and TVD_RS00880 (TvSDMT) after addition of 0.05 mM IPTG. Cell extracts were prepared by high pressure homogenization in buffer A (20 mM Tris–HCl, 300 mM NaCl, 20 mM imidazole, 2 mM DTT, pH 7.5). His-tagged proteins were purified by an affinity column packed with Ni Sepharose (Cytiva, Uppsala, Sweden). After the pretreated sample loaded onto the affinity column was washed with 10 column volumes of buffer A and then 5 column volumes of 5% buffer B (20 mM Tris–HCl, 300 mM NaCl, 44 mM imidazole, 2 mM DTT, pH 7.5), TvGMT or TvSDMT was eluted using 30% buffer B (20 mM Tris–HCl, 300 mM NaCl, 164 mM imidazole, 2 mM DTT, pH 7.5). Protein concentrations were determined by Bradford assay using BSA as standard. The purities of TvGMT and TvSDMT were examined based on SDS-PAGE analysis with Coomassie staining.