4.6 Article

Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents

Journal

PROTEIN SCIENCE
Volume 20, Issue 6, Pages 1030-1041

Publisher

WILEY-BLACKWELL
DOI: 10.1002/pro.636

Keywords

cell-free; family B GPCR; functional ligand binding; CRFR; integral membrane protein; polymer; NMR; static light scattering

Funding

  1. National Institute of Diabetes and Digestive and Kidney Diseases [5P01DK026741-30]
  2. National Institute of General Medical Sciences [U54 GM074929]
  3. Clayton Medical Research Foundation, Inc.
  4. NIH
  5. IFEZ (Incheon)
  6. WCU-Korea Programs
  7. Deutsche Forschungs Gemeinschaft (DFG)
  8. National Research Foundation of Korea [R32-2011-000-10215-0] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)

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G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2 beta are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.

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