Figure 3.

EF-Tu can be site-specifically cleaved in a purified three-component system. Purified His-tagged EF-Tu (2 μM) was incubated in the presence or absence of Lit (1 μM) and the synthetic gol peptide (5 μM) in 50 mM Tris⋅HCl, pH 8.0 containing 10 mM 2-mercaptoethanol, 5 mM MgCl₂, 10 μM GDP, and 30 μM sarkosyl at 37°C for 1 hr. Samples were then boiled in SDS sample buffer, resolved on a 12% SDS-polyacrylamide gel, and stained with Coomassie blue. Lanes: 1, no Gol peptide; 2, no Lit protein; 3, all three components; 4, all three components but with EDTA added to 10 mM before incubation.