Heterologous expression of membrane proteins in Escherichia coli often requires optimization to overcome problems with toxicity of the recombinant protein to the host cell. A number of Gateway-based destination vectors were constructed to investigate expression of membrane proteins using a high-throughput approach. These vectors were tested using putative drug transporter proteins from the multidrug and toxic compound extrusion (MATE) family and the resistance-nodulation-cell division superfamily encoded by the human pathogen Acinetobacter baumannii. Active transport of antibiotics and antiseptics mediated by efflux proteins contributes to the high level of multidrug resistance observed in A. baumannii. Substrates for 4 of the 5 putative efflux proteins investigated were identified using the expression vectors designed in this study. Additionally, a Gateway-based suicide vector was designed for construction of specific A. baumannii insertion disruption mutants. This knockout cloning strategy was tested and shown to be successful in inactivating AbeM4, a putative MATE family protein. Therefore, we have shown that the Gateway-based vectors constructed in this study are versatile tools that can be used for manipulation and characterization of membrane proteins.