Abstract
The 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.
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Abbreviations
- AMPA and NMDA:
-
Ionotropic glutamate receptors
- ATP:
-
Adenosine triphosphate
- cAMP:
-
Cyclic AMP
- cDNA:
-
Complementary DNA
- CREB:
-
cAMP-response element-binding protein
- CTP:
-
Cytidine triphosphate
- dNTP:
-
deoxyribonucleotide triphosphate
- D2L :
-
Dopamine D2 receptor long isoform
- D2S :
-
Dopamine D2 receptor short isoform
- DARPP-32:
-
32kDa dopamine and cAMP-regulated phosphoprotein
- DNA:
-
Deoxyribonucleic acid
- E. coli:
-
Escherichia coli
- ECL:
-
Enhanced chemiluminescence
- EDTA:
-
Ethylenediaminetetraacetic acid
- GPCR:
-
G-protein-coupled receptor
- Gαs:
-
Stimulatory G-protein
- GTP:
-
Guanosine triphosphate
- HRP:
-
Horseradish peroxidase
- LB:
-
Lysogeny broth
- MOBIX:
-
McMaster Institute for Molecular Biology and Biotechnology
- Ni–NTA:
-
Nickel–nitrilotriacetic acid
- NPA:
-
Norpropylapomorphine
- PAGE:
-
Polyacrylamide gel electrophoresis
- PBS:
-
Phosphate buffered saline
- PCR:
-
Polymerase chain reaction
- PKA:
-
Protein kinase A
- PVDF:
-
Polyvinylidene fluoride
- RNA:
-
Ribonucleic acid
- SDS:
-
Sodium dodecyl sulfate
- SDS:
-
Tris-acetate-EDTA buffer
- UTP:
-
Uridine triphosphate
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The study was funded by the National Institutes of Health, Canadian Institutes of Health Research, and the Ontario Mental Health Foundation.
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Basu, D., Daya, R., Sookram, C.D.R., Mishra, R.K. (2015). Cell-Free Protein Synthesis and Purification of the Dopamine D2 Receptor. In: Tiberi, M. (eds) Dopamine Receptor Technologies. Neuromethods, vol 96. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2196-6_4
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DOI: https://doi.org/10.1007/978-1-4939-2196-6_4
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