Expression, purification and reconstitution of the 4-hydroxybenzoate transporter PcaK from Acinetobacter sp. ADP1

Protein Expr Purif. 2014 Sep;101(100):68-75. doi: 10.1016/j.pep.2014.05.011. Epub 2014 Jun 5.

Abstract

The aromatic acid:H(+) symporter family of integral membrane proteins play an important role in the microbial metabolism of aromatic compounds. Here, we show that the 4-hydroxybenzoate transporter from Acinetobacter sp. ADP1, PcaK, can be successfully overexpressed in Escherichia coli and purified by affinity chromatography. Affinity-purified PcaK is a stable, monodisperse homotrimer in the detergent n-dodecyl-β-d-maltopyranoside supplemented with cholesteryl hemisuccinate. The purified protein has α-helical secondary structure and can be reconstituted to a functional state in synthetic proteoliposomes. Asymmetric substrate transport was observed when proteoliposomes were energized by applying an electrochemical proton gradient (Δμ‾H(+)) or a membrane potential (ΔΨ) but not by ΔpH alone. PcaK was selective in transporting 4-hydroxybenzoate and 3,4-dihydroxybenzoate over closely related compounds, confirming previous reports on substrate specificity. However, PcaK also showed an unexpected preference for transporting 2-hydroxybenzoates. These results provide the basis for further detailed studies of the structure and function of this family of transporters.

Keywords: Enzyme purification; Membrane proteins; Membrane transport; Recombinant protein expression; Reconstitution of membrane transporters.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acinetobacter / enzymology*
  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Biological Transport / physiology
  • Carrier Proteins / biosynthesis
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Cholesterol Esters / chemistry
  • Chromatography, Affinity
  • Drug Resistance, Bacterial / physiology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Hydroxybenzoates / metabolism
  • Maltose / analogs & derivatives
  • Maltose / chemistry
  • Parabens / metabolism*
  • Protein Structure, Secondary
  • Proteolipids / chemistry
  • Proteolipids / metabolism
  • Salicylic Acid / metabolism
  • Substrate Specificity
  • Symporters / biosynthesis
  • Symporters / genetics*
  • Symporters / metabolism

Substances

  • 3,4-dihydroxybenzoate
  • Bacterial Proteins
  • Carrier Proteins
  • Cholesterol Esters
  • Hydroxybenzoates
  • Parabens
  • Proteolipids
  • Symporters
  • dodecyl maltopyranoside
  • proteoliposomes
  • Maltose
  • 4-hydroxybenzoic acid
  • Salicylic Acid
  • cholesteryl succinate