Expression, purification, and characterization of the carboxyl-terminal region of the Na⁺/H⁺ exchanger

The Na⁺/H⁺ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na⁺. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S- transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na⁺/H⁺ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S- transferase and the Na⁺/H⁺ exchanger domain. Free Na⁺/H⁺ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% α-helix, 17% β-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.