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January 15, 2009 (Vol. 29, No. 2)

Tools for Studying Membrane Protein Targets

Technology Useful for High-Throughput Screening and Lead-Optimization Projects

  • Lipoparticles

    Click Image To Enlarge +
    Figures: 1A, 1B and 1C

    A novel format for the experimental manipulation of membrane proteins, and one that overcomes the restrictions of existing approaches, is Lipoparticle technology from Integral Molecular. Lipoparticles are virus-like particles that present high concentrations of membrane protein on their surfaces. Because embedded membrane proteins are captured directly from mammalian cell surfaces, their native structure is preserved without the need for detergents or fusion partners. This technology, which can be used to isolate any plasma membrane protein, results in a nanoparticulate suspension of the target protein that can be utilized in diverse assays where the use of cells or membrane preparations is limited.

    Lipoparticles are produced from mammalian cells by co-expressing the retroviral structural core polyprotein Gag, along with a desired membrane protein. Each protein traffics to the cell membrane, where Gag proteins self assemble into core capsid structures which bud through the plasma membrane, capturing target membrane proteins (Figure 1A).

    Because Lipoparticle formation is an active process mediated by Gag, the resulting particles are morphologically homogeneous. Dynamic light scattering and electron microscopy confirm they are free of contaminating cellular debris (Figure 1B). With a mean diameter of approximately 150 nm and a narrow size distribution (Figure 1C), they are readily suspended in aqueous buffers.

    Radioligand binding assays using Lipoparticle suspensions confirm the functional integrity of incorporated membrane proteins, as shown for Lipoparticles containing the chemokine receptor CXCR4 (Figure 2A). Binding of the CXCR4 ligand, SDF-1a, is specific, concentration-dependent, and saturable, exhibiting a low nanomolar KD. Its similarities to cell-bound receptor behavior confirm that the Lipoparticle-incorporated receptor is structurally and functionally intact.

    The CXCR4 competitive binding curve indicates an estimated Lipoparticle receptor content of approximately 200 pmol/mg total protein, which is approximately 20–100 fold higher than what is found in conventional membrane preparations. Similar receptor concentrations, typically between 50–200 pmol/mg, have been determined for a diverse range of other membrane proteins incorporated into Lipoparticles.

    The high concentration of membrane proteins in Lipoparticles enables their use for rapid screening of membrane protein interactions in conventional and nonconventional formats. The binding of a diverse panel of antibodies with specificity for the chemokine receptor CCR5 was assessed using ELISA in 96-well microplates. When Lipoparticles incorporating CCR5 were included in the plates, high levels of binding were observed for all antibodies, including several (45523, 45529, and 45517) that recognize complex, nonlinear epitopes (Figure 2B).

    None of the antibodies recognized Lipoparticles incorporating a different chemokine receptor (CXCR4). The highly concentrated, homogeneous membrane proteins contained in Lipoparticle suspensions enable drug and antibody screening in high-throughput formats that have previously been convenient only for soluble proteins.

  • Click Image To Enlarge +
    Figures: 2A and 2B

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