Fig. 4.

Functional complementation of E14C-His by EmrE. E14C-His (50 ng of DNA) and EmrE (300 ng of DNA) were coexpressed in vitro. After the synthesis reaction was completed, DDM was added to 0.08%, and the reaction was loaded on Ni-NTA beads. The protein that bound to beads was washed twice with 0.08% DDM/Na buffer, and samples (▴) were assayed for TPP⁺-binding activity, as described in Experimental Procedures. TPP⁺-binding activity was measured also for E14C-His (▪) that was synthesized in vitro in a separate reaction. TPP⁺ concentration used for binding was 5 nM for E14C-His/EmrE and 20 nM for the inactive mutant E14C-His alone.