The in vitro interaction of CmeA with CmeC

The membrane fusion protein CmeA and the outer membrane channel CmeC, two important components of CmeABC system in Campylobacter jejuni, were expressed in Escherichia coli. After Ni-NTA and ion exchange columns purification, size exclusion chromatography showed that CmeA primarily existed as trimer and CmeC existed as monomer. Then the interaction between CmeA and CmeC was analyzed by dithiobis (succinimidyl propionate) (DSP) chemical crosslinking, pull-down assay on a Ni-NTA column, and isothermal titration calorimetry (ITC) measurement. The results clearly showed the CmeA–CmeC complex band, which confirmed the interaction in vitro and this interaction is independent of substrate and CmeB. It suggests that the mechanism underlying CmeABC function in C. jejuni is similar to that of AcrAB-TolC in E. coli.