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% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.
Gebreselassie
D
D
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Rajarathnam
K
K
Fliegel
L
L
D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry; D005821:Genetic Techniques; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers