Doxorubicin transport measurements for a bacterial multidrug transport protein.

Multidrug transporters are membrane proteins that confer antibiotic resistance on bacterial cells by actively extruding toxic compounds. NorM from Vibrio cholerae (NorM-VC) belongs to the MATE (Multidrug And Toxic compound Extrusion) family and is active as an ion/antibiotic exchanger. With the availability of high-resolution crystal and cryo-EM structures, and mechanistic predictions for NorM proteins, there is a need for convenient activity measurements in non-pathogenic cells and cell mimics. Here, we outline methods for the expression and purification of NorM-VC, protein reconstitution in proteoliposomes, and transport activity measurements in Lactococcus lactis and proteoliposomes using doxorubicin as the substrate. These measurements can easily be adapted for high throughput measurements in multi-well plates and extended to other classes of multidrug transporters that use doxorubicin as a substrate.