Mms 156p

T. cruzi genes, cloned into pET21a, were expressed with a C-terminal His₆ tag in BL21-DE3 Escherichia coli grown in Luria broth after induction with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 1 h. The tagged proteins were purified using Ni-NTA Superflow beads (Qiagen). Purified proteins were diluted 1:100 in 20 mM NaCl, 10 mM Tris, pH 7.5 before adding GzmB at the indicated concentrations. Reactions were stopped after 30 min by boiling in SDS-PAGE loading buffer. Samples were analyzed by immunoblot using anti–6-His mouse monoclonal antibody (Covance, MMS-156P).

Bacterial genes, expressed from pET21a with a C-terminal His₆ tag in BL21-DE3 Ec grown in Luria broth, were induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma) for 1 h. The tagged proteins were purified using Ni-NTA Superflow beads (Qiagen). Purified proteins were diluted 1:100 in 20 mM NaCl, 10 mM TrisHCl, pH 7.5, before adding GzmB at the indicated concentration. To assess intracellular cleavage, GzmB and sublytic GNLY were added to Ec expressing His-tagged aaRS that were induced for 30 min at 37°C with 0.1 mM IPTG, in 50 mM NaCl, 10 mM TrisHCl, pH 7.4. Reactions were stopped after 30 min by boiling in SDS-PAGE loading buffer. Samples were analyzed by immunoblot using anti-His₆ mouse monoclonal Ab (Covance, MMS-156P). As a loading control, blots were probed for cytochrome C (Abcam, ab18738).