Expression and purification of the intact bacterial ergothioneine transporter EgtU

The bacterial ATP-binding cassette (ABC) transporter EgtU is responsible for uptake of the cellular antioxidant ergothioneine in Streptococcus pneumoniae, and it has homologs in a surprisingly diverse range of microbial pathogens. Crystal structures have been reported for the solute binding domain of EgtU, but many details of the structure and function of the intact heterotetrameric transporter remain to be elucidated. In this study, we have expressed S. pneumoniae EgtU and purified it from E. coli BL21 (DE3) with high purity and homogeneity. Our preliminary data establish ergothioneine binding and ATP hydrolysis by the full-length transporter solubilized in DDM micelles. Our workflow allows for isolation of suitable quantities of EgtU for ongoing structural studies and detailed biophysical characterization. •The bacterial ABC transporter EgtU imports ergothioneine.•EgtUBC, the transmembrane component of the EgtU transporter, was coexpressed with EgtUA, the nucleotide binding domain.•EgtUBC was solubilized and copurified with EgtUA.•This study will structural and mechanistic analysis of the EgtU transporter.