Cell-Free Protein Synthesis and Purification of the Dopamine D2 Receptor

Abstract

AbstractThe dopamine D2 receptor is considered one of the most important neurotransmitter receptors relevant to behavioral and clinical effects of antipsychotic drugs. Its expression and purification however is met with several challenges. This chapter provides a detailed methodology on the cell-free synthesis of the dopamine D2L receptor, using Escherichia coli (E. coli) lysate in a regenerative dialysis membrane system. This cell-free technique utilizes protein synthesis machinery and exogenous dopamine D2L DNA to synthesize functional protein outside of intact cells. The cell-free system offers various advantages specifically for the expression of transmembrane proteins, like G-protein-coupled receptors, which typically present a significant challenge. Transmembrane protein synthesis via more conventional approaches exhibit a number of innate limitations including protein aggregation, misfolding, and low yield due to cellular toxicity. The cell-free protein synthesis systems allow for the continuous replenishment of depleting precursors and removal of toxic buildup through a size-regulated porous dialysis membrane. As such this system facilitates higher yields of G-protein-coupled receptors when compared to conventional cell-based methods. Furthermore, this method provides the capability to modify the protein product, as it can be designed to incorporate radiolabeled isotopes, unnatural amino acids, solubilizing agents, cofactors, and inhibitors as is relevant for more innovative and specific research questions. Finally, an optimized cell-free system can synthesize high levels of this G-protein-coupled receptor within a few hours of incubation, providing an efficient solution to the challenge of characterizing the dopamine D2 receptor.Key wordsCell-free protein synthesis E. coli Dopamine D2 receptorGPCRTransmembrane protein