*5.1. Expression and Purification of MTase*

The histidine-tagged MTase (MTase) gene of approximately 960 bases coding for 33–353 amino acids of genotype 1 (GenBank accession no. AF444002.1) was cloned in pET 28a (+) vector (GenScript, New Jersey, USA) and confirmed by restriction digestion using NdeI and XhoI enzymes. The positive pET28a (+)-MTase construct was transformed into BL21 (DE3) *E. coli* cells to express the protein. A single positive clone carrying the MTase gene was grown in LB broth, and the logarithmic phase culture was induced with 1 mM IPTG at 37 ◦C for 3 h. The induced culture was centrifuged at 5000× *g* for 20 min at 4 ◦C, and the pellet was suspended in lysis buffer (10 mM Tris–Cl pH 8.0, 1 mM EDTA, 100 mM

NaCl, 5 mM DTT, and 500 µg/mL lysozyme) and sonicated for 5 min (10 s on, 20 s off). The cell lysate was centrifuged for 15,000× *g* at 4 ◦C for 60 min. The pellet was further resuspended in 20 mL IB (inclusion bodies) wash buffer (10 mM Tris–Cl pH 8.0, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100) by continuous stirring for 2 h and centrifuged at 15,000× *g* for 45 min at 4 ◦C. The pellet obtained in IBs was solubilised in buffer (10 mM Tris–HCl pH 8.0, 100 mM NaCl, and 0.5% NLS) (N-lauryl sarcosine, Sigma Aldrich, Burlington, MA, USA) by continuous stirring for overnight at 4 ◦C. The solubilised pellet was centrifuged at 15,000× *g* for 45 min at 4 ◦C and subsequently filtered through a 0.45 µM syringe filter (Millipore). The solubilised protein was purified using metal (Ni-NTA) affinity chromatography using AKTA start (GE Healthcare, Chicago, IL, USA). The protein was eluted in 10 mM Tris–Cl, pH 8.0, 100 mM NaCl, 0.01% NLS, and 200 mM imidazole. The purified MTase was further characterised by Western blot analysis, as described previously [33,34]. The membrane was probed with MTase epitope-specific primary antibody followed by HRP-conjugated goat anti-rabbit secondary antibody (Invitrogen, Waltham, MA, USA). Signal was detected using electrochemiluminescence (Bio-Rad Western ECL substrate, Hercules, CA, USA).
