Biosynthesis and purification of a hydrophobic peptide from transmembrane domains of G-protein-coupled CB2 receptor

Authors

  • H. Zheng,

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author
  • J. Zhao,

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author
  • S. Wang,

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author
  • C.-M. Lin,

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author
  • T. Chen,

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author
  • D.H. Jones,

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author
  • C. Ma,

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author
  • S. Opella,

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author
  • X.-Q. Xie

    1. H. Zheng, J. Zhao and X.-Q. Xie, Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, USA
      H. Zheng, J. Zhao, S. Wang and X.-Q. Xie, Department of Pharmaceutical Science, University of Connecticut, Storrs, CT, USA
      C.-M. Lin and T. Chen, Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT, USA
      D.H. Jones, C. Ma and S. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
    Search for more papers by this author

  • Present address: H. Zheng, Department of Basic and Pharmaceutical Sciences, Albany College of Pharmacy, Albany, NY, USA.

Xiang-Qun (Sean) Xie
Department of Pharmaceutical & Pharmacological Sciences
College of Pharmacy
University of Houston
Houston
TX77204-5037
Tel.: 713-743-1288
Fax: 713-743-1229
E-mail: xxie@uh.edu

Abstract

Abstract:  A major challenge for the structural study of the seven-transmembrane G-protein-coupled receptors is to obtain a sufficient amount of purified protein at the milligram level, which is required for either nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. In order to develop a high-yield and cost-effective method, and also to obtain preliminary structural information for the computer modeling of the three-dimensional receptor structural model, a highly hydrophobic peptide from human cannabinoid subtype 2 receptor CB265−101, was chosen to develop high-yield membrane protein expression and purification methods. The peptide included the second transmembrane helix with the associated loop regions of the CB2 receptor. It was over-expressed in Escherichia coli, with a modified TrpΔ LE1413 (TrpLE) leading fusion sequence and a nine-histidine tag, and was then separated and purified from the tag in a preparative scale. An experimental protocol for the chemical cleavage of membrane protein fragment was developed using cyanogen bromide to remove the TrpLE tag from the hydrophobic fusion protein. In addition, protein uniformly labeled with isotopic 15N was obtained by expression in 15N-enriched minimum media. The developed and optimized preparation scheme of expression, cleavage, and purification provided a sufficient amount of peptide for NMR structure analysis and other biophysical studies that will be reported elsewhere. The process of fusion protein cleavage following purification was monitored by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), and the final sample was validated by MS and circular dichroism experiments.

Ancillary