Fig 2: Purification of RNase I (6×His) and RNase activity assay. (A) Purification of RNase I (6×His) from Nickel-NTA agarose column. Lane 1, RNase I (6×His) pooled fractions from a nickel column (purified from T7 Express cell extract). Arrows indicate the cytoplasmic RNase I precursor (cRNase I) with the signal peptide (predicted MW 30.7 kDa), and the periplasmic RNase I with the signal peptide removed (predicted MW 27.0 kDa). (B) RNase activity on a FAM-labeled SARS-CoV-2 RNA (50 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase If (NEB). RNase I (6×His) enzyme titration (2 µg to 25 ng protein) was used in the activity assay to digest fixed amount of RNA (16 nM) in NEB buffer 3 at 37°C for 1 h. Proteinase K (1.6 U) was added to remove RNase I. The final cleavage products were analyzed by capillary electrophoresis (CE), and peaks were visualized by PeakScan.