Figure 7
Characterization of the Cys-less CLLD mutant. (a) Gel-filtration profile of the mutant. V0 and Vt indicate the void and total volumes of the column, respectively. The elution volume is 74.8 ml. (b) Coomassie Blue-stained SDS–PAGE of the protein with the amount loaded indicated at the top of each lane in µg. Molecular-weight standards are included in the left lane (labelled in kDa). The mutant runs at ∼27 kDa, as observed for CLLD DgkA, suggesting that they share the same oligomeric state (Li, Shah et al., 2013). (c) Progress curve of the coupled kinase reaction recorded in the absence of protein (dotted line) and in the presence of CLLD (solid line) and the Cys-less mutant (dashed line). 9.9 MAG was used as the host lipid as well as the lipid substrate for the in meso assay of DgkA (Li & Caffrey, 2011). |