期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 101, 期 -, 页码 68-75出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2014.05.011
关键词
Enzyme purification; Recombinant protein expression; Membrane proteins; Membrane transport; Reconstitution of membrane transporters
类别
资金
- European Research Council under the European Union's Seventh Framework Programme (FP7) [282101]
- Engineering and Physical Sciences Research Council Doctoral Training Centre Grant [EP/G036780/1]
- European Research Council (ERC) [282101] Funding Source: European Research Council (ERC)
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-beta-D-maltopyranoside supplemented with cholesteryl hemisuccinate. The purified protein has alpha-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 (Delta(mu) over bar (+)(H)) or a membrane potential (Delta Psi) but not by Delta pH 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. (C) 2014 The Authors. Published by Elsevier Inc.
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