Functional expression of a two-transmembrane HtrII protein using cell-free synthesis

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Author(s)

    • Sudo Yuki
    • Division of Biological Science, Graduate School of Science, Nagoya University|PRESTO, Japan Science and Technology Agency (JST)
    • Kamo Naoki
    • College of Pharmaceutical Sciences, Matsuyama University
    • Kojima Chojiro
    • Graduate School of Biological Sciences, Nara Institute of Science and Technology|Institute for Protein Research, Osaka University

Abstract

An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the <i>pharaonis</i> halobacterial transducer protein, <i>p</i>HtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescent protein and maltose binding protein). In this system, all fusion <i>p</i>HtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-<font size=-2>D</font>-maltoside was added for enhancing the solubilization of the hydrophobic region of <i>p</i>HtrII. The activity of the expressed <i>p</i>HtrII, having various tags, was checked using a pull-down assay, using the fact that <i>p</i>HtrII forms a signaling complex with <i>pharaonis</i> phoborhodopsin (<i>p</i>pR) in the membrane, as also in the presence of a detergent. All tagged <i>p</i>HtrII showed a binding activity with <i>p</i>pR. Interestingly, the binding activity with <i>p</i>pR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.<br>

Journal

  • BIOPHYSICS

    BIOPHYSICS (7), 51-58, 2011

    The Biophysical Society of Japan

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