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 Citations (Scopus)

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 - 1998
Externally publishedYes

Fingerprint

Sodium-Hydrogen Antiporter
Purification
Proteins
Inclusion Bodies
Glutathione Transferase
Electrophoresis
Fusion reactions
Secondary Protein Structure
Circular dichroism spectroscopy
Dialysis
Circular Dichroism
Thrombin
Cell membranes
Cell culture
Protons
Blood Proteins
Polyacrylamide Gel Electrophoresis
Spectrum Analysis
Escherichia coli
Buffers

Keywords

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

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger. / Gebreselassie, Daniel; Rajarathnam, Krishna; Fliegel, Larry.

In: Biochemistry and Cell Biology, Vol. 76, No. 5, 1998, p. 837-842.

Research output: Contribution to journalArticle

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