Expression and Characterization of E. coli YchM, a Bacterial Member of the SLC26 Family of Anion Transporters

Abstract

The widely-expressed members of the SLC26 family of anion transporters play a crucial role in the transport of bicarbonate, chloride, sulfate and iodide in epithelial cells and mutations cause a number of serious human genetic disorders including cystic fibrosis, dystropic dysplasia, congenital chloride diarrhea and deafness. To elucidate the molecular structure of SLC26 transporters and their mechanism of action, the E. coli homologue, YchM transporter, was chosen for analysis. YchM consists of two functional domains: the N-terminal membrane domain responsible for transport activity, and the C-terminal STAS domain, whose crystal structure resembles SpoIIAA, the anti-sigma factor antagonist from Bacillus. YchM and its membrane domain with a C-terminal His-tag were expressed in various E. coli strains (Tuner, C41, C43) grown at 20°C and purified using Ni-NTA affinity chromatography and gel filtration. Stability tests were carried out with a range of detergents, with n-dodecyl-β-D-maltopyranoside (DDM) at pH 6.0, 100mM NaCl and 2% glycerol being optimal. The CD-spectrum of YchM revealed a high helical content, and the thermal titration profile revealed that YchM protein has 2-step titration curve at 40°C and 55°C. YchM was prone to aggregation while sparce-matrix screening of the membrane domain gave small crystals that diffracted to ∼12Å. Further refinement of the purification and crystallization conditions are underway. Supported by operating and training grants from CIHR.