Jump to main content
Jump to site search


One-step methodology for the direct covalent capture of GPCRs from complex matrices onto solid surfaces based on the bioorthogonal reaction between haloalkane dehalogenase and chloroalkanes

Author affiliations

Abstract

Protein immobilization techniques play an important role in the development of assays for disease diagnosis and drug discovery. However, many of these approaches are not applicable to transmembrane proteins. G protein-coupled receptors (GPCRs) are the largest protein superfamily encoded by the human genome and are targeted by a quarter of all prescription drugs. GPCRs are highly dynamic and sensitive to changes in the ambient environment, and current immobilization methodologies are not suitable for GPCRs. We used haloalkane dehalogenase (Halo) as an immobilization tag fused to the β2-adrenoceptor (β2-AR), angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors. The engineered Halo-tag covalently binds to a specific substrate chloroalkane through Asp 106 in the catalytic pocket. The Halo-tagged GPCRs were expressed in Escherichia coli at a suitable yield. Accordingly, we loaded cell lysate containing Halo-tagged GPCRs onto a macroporous silica gel coated with chloroalkane. Morphological characterization indicated a homogeneous monolayer of immobilized Halo-tagged GPCRs on the silica gel surface. The immobilized receptors proved to be surrounded by specific bound phospholipids including PG C18:1/C18:1. We observed a radio-ligand binding ability and ligand-induced conformational changes in the immobilized GPCRs, suggesting the preservation of bioactivity. This method is a one-step approach for the specific immobilization of GPCRs from cell lysates and validates that immobilized receptors retain canonical ligand binding capacity. Our immobilization strategy circumvents labor-intensive purification procedures and minimizes loss of activity. The immobilized receptors can be applied to high-throughput drug and interaction partner screening for GPCRs.

Graphical abstract: One-step methodology for the direct covalent capture of GPCRs from complex matrices onto solid surfaces based on the bioorthogonal reaction between haloalkane dehalogenase and chloroalkanes

Back to tab navigation

Supplementary files

Publication details

The article was received on 05 Sep 2017, accepted on 18 Oct 2017 and first published on 19 Oct 2017


Article type: Edge Article
DOI: 10.1039/C7SC03887A
Citation: Chem. Sci., 2018, Advance Article
  • Open access: Creative Commons BY license
  •   Request permissions

    One-step methodology for the direct covalent capture of GPCRs from complex matrices onto solid surfaces based on the bioorthogonal reaction between haloalkane dehalogenase and chloroalkanes

    K. Zeng, Q. Li, J. Wang, G. Yin, Y. Zhang, C. Xiao, T. Fan, X. Zhao and X. Zheng, Chem. Sci., 2018, Advance Article , DOI: 10.1039/C7SC03887A

    This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material.

    Reproduced material should be attributed as follows:

    • For reproduction of material from NJC:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
    • For reproduction of material from PCCP:
      [Original citation] - Published by the PCCP Owner Societies.
    • For reproduction of material from PPS:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
    • For reproduction of material from all other RSC journals:
      [Original citation] - Published by The Royal Society of Chemistry.

    Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.

Search articles by author

Spotlight

Advertisements