Cell‐Free Expression of G Protein–Coupled Receptors

Abstract

The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes.

Membranes under the Magnetic Lens: A Dive into the Diverse World of Membrane Protein Structures Using Cryo-EM

“…The feasibility of this technique for mammalian cells still needs to be established. Moreover, developments in cell-free expression systems have resulted in membrane proteins, such as GPCRs, being produced outside of the cell, and interested readers can follow up on recent developments in published reviews and papers. − Although this approach has not yet yielded high resolution cryo-EM structures, proteins produced in this way have the potential to impact the cryo-EM field in the future.…”

Synthetic Biology Goes Cell-Free

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Chaperonin-enhanced Escherichia coli cell-free expression of functional CXCR4

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