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

Daniel Gebreselassie, Krishna Rajarathnam, Larry Fliegel

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)837-842
Number of pages6
JournalBiochemistry and Cell Biology
Volume76
Issue number5
DOIs
StatePublished - Dec 1 1998
Externally publishedYes

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Keywords

  • Circular dichroism
  • Membrane protein
  • Na/H exchanger
  • pH regulation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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